Method of treating or preventing stroke

ABSTRACT

A method of reducing an effect of stroke in a subject, the method comprising administering to the subject a compound that inhibits VEGF-B signaling

RELATED APPLICATION DATA

The present application claims priority from Australian PatentApplication No. 2014904606 entitled “Method of Treating or PreventingStroke” filed on 17 Nov. 2014. The entire contents of which are herebyincorporated by reference.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicform. The entire contents of the Sequence Listing are herebyincorporated by reference.

FIELD

The present disclosure relates to methods of treating or preventing theeffects of stroke in a subject by antagonizing vascular endothelialgrowth factor (VEGF)-B.

BACKGROUND

Stroke is the second leading cause of mortality after heart disease andthe leading cause of disability in Australia. It is the third leadingcause of death in the United States, with over 140,000 people dying eachyear from stroke. It is also the leading cause of serious, long termdisability in the United States. Projected costs for stroke in USA forthe period from 2005 to 2050 are US$2.2 trillion.

Disability affects 75% of stroke survivors enough to decrease theiremployability. Stroke can affect subjects physically, mentally,emotionally, or a combination of the three.

Some of the physical disabilities that can result from stroke includemuscle weakness, numbness, pressure sores, pneumonia, incontinence,apraxia (inability to perform learned movements), difficulties carryingout daily activities, appetite loss, speech loss, vision loss, and pain.If the stroke is severe enough, or in a certain location such as partsof the brainstem, coma or death can result.

Emotional problems resulting from stroke can result from direct damageto emotional centers in the brain or from frustration and difficultyadapting to new limitations. Post-stroke emotional difficulties includedepression, anxiety, panic attacks, flat affect (failure to expressemotions), mania, apathy, and psychosis.

Cognitive deficits resulting from stroke include perceptual disorders,speech problems, dementia, and problems with attention and memory. Astroke sufferer may be unaware of his or her own disabilities, acondition called anosognosia. In a condition called hemispatial neglect,a patient is unable to attend to anything on the side of space oppositeto the damaged hemisphere.

Up to 10% of all stroke patients develop seizures, most commonly in theweek subsequent to the event. The severity of the stroke increases thelikelihood of a seizure.

Stroke is the rapidly developing loss of brain function(s) due todisturbance in the blood supply to the brain. This can be due toischemia (lack of blood flow) caused by blockage (thrombosis, arterialembolism), or a hemorrhage (leakage of blood). As a result, the affectedarea of the brain is unable to function, which might result in asubject's inability to move one or more limbs on one side of the body,inability to understand or formulate speech, or an inability to see oneside of the visual field. Stroke often results in neuronal cell deathand can lead to death.

There are two common types of stroke: (i) ischemic stroke, which iscaused by a temporary or permanent occlusion to blood flow to the brain,and accounts for 85% of stroke cases, and (ii) hemorrhagic stroke, whichis caused by a ruptured blood vessel and accounts for the majority ofthe remaining cases. The most common cause of ischemic stroke isocclusion of the middle cerebral artery (the intra-cranial arterydownstream from the internal carotid artery), which damages cerebrum(e.g., cerebral cortex), e.g., the motor and sensory cortices of thebrain. Such damage results in hemiplegia, hemi-anesthesia and, dependingon the cerebral hemisphere damaged, either language or visuo-spatialdeficits.

Some neuroprotective agents have been tested for efficacy in treatmentof stroke, and have failed, including N-methyl-D-aspartate receptorantagonists including lubeluzole), nalmefene, clomethiazole, calciumchannel blockers (includinga-amino-3-hydroxy-5-rnethylisoxazole-4-proprionic acid antagonists,serotonin agonists (e.g., repinotan), and transmembrane potassiumchannel modulators), tirilazad, anti-ICAM-I antibody, humanantileukocytic antibody (Hu23F2G), antiplatelet antibody (e.g.,abciximab), citicoline (an exogenous form ofcytidine-5′-diphosphocholine), and basic fibroblast growth factor.

It will be apparent from the foregoing that there is a need in the artfor therapeutics for stroke.

The growth factor VEGF-B has also been studied for an effect in neuronalsurvival, including after a stroke. Sun et al., J. Cereb. Blood Flow andMetab., 24: 1146-1152, 2004 studied stroke in mice lacking VEGF-B andfound that the lack of this growth factor resulted in significantlylarger infarct volume and neurologic impairment. Li et al., J Clin.Invest., 118: 913-923, 2008 showed that injection of VEGF-B into thebrain of mice in which a stroke has been induced rescued neurons fromapoptosis, again suggesting a role for this growth factor in providingtherapeutic benefit following stroke. Extending these studies, Li etal., Cell Adhesion and Migration, 3: 322-327, 2009 suggested that VEGF-Bprotected neurons against apoptosis and mused that this growth factormay have therapeutic value in treating neurodegenerative diseases.

SUMMARY

In producing the present invention, the inventors studied the effects ofinhibiting signaling of VEGF-B in an accepted mouse model of stroke,e.g., ischemic stroke. The inventors studied the effect of this growthfactor by administering an antagonist of VEGF-B (e.g., an antagonisticantibody) before or after induction of stroke. Counter to what wasimplied by the studies discussed above, the inventors were able toreduce an effect of stroke, e.g., infarct size, intracerebral hemorrhagescore or blood brain barrier breakdown. This is distinct from preventingthe occurrence of a stroke. The inventors were also able to improveoutcomes of thrombolytic therapy of stroke, e.g., by reducing infarctsize, hemorrhages (e.g., intracerebral hemorrhages) and the number oflethal hemorrhages. The inventors also showed that administering anantagonist of VEGF-B (e.g., an antagonistic antibody) after strokeextended the time in which a thrombolytic agent could safely beadministered to a subject.

The findings by the inventors provide the basis for methods for reducingthe effects of stroke in a subject by inhibiting VEGF-B signaling. Thefindings also provide the basis for methods of prophylaxis or treatmentof the effects of stroke in a subject by inhibiting VEGF-B signaling.

For example, the present disclosure provides a method of reducing aneffect of stroke in a subject, the method comprising administering tothe subject a compound that inhibits VEGF-B signaling.

The inventors have also found that they can reduce the incidence ofhemorrhage in a subject who has suffered a stroke. Accordingly, thepresent disclosure additionally provides a method for reducing theincidence of hemorrhage in a subject who has suffered a stroke, themethod comprising administering a compound that inhibits VEGF-Bsignaling. In one example, the method additionally comprisesadministering a thrombolytic compound.

This finding also provides the basis for methods for continuing to treator commencing to treat a subject who has suffered a stroke to prevent ahemorrhage, e.g., an intracerebral hemorrhage, the method comprisingadministering a compound that inhibits VEGF-B signaling. For example, asubject can be retreated with a compound that inhibits VEGF-B signalingafter a performing a method disclosed herein to reduce the risk of or toprevent a hemorrhage, e.g., an intracerebral hemorrhage.

The inventors have also found that they can reduce the likelihood oflethal hemorrhage in a subject who has suffered a stroke. Accordingly,the present disclosure additionally provides a method for reducing thelikelihood of lethal hemorrhage in a subject who has suffered a stroke,the method comprising administering a compound that inhibits VEGF-Bsignaling. In one example, the method additionally comprisesadministering a thrombolytic compound.

The inventors also found that they could reduce blood-brain-barrierbreakdown or leakage. Thus, the inventors have also provided a methodfor preventing blood-brain-barrier breakdown or leakage, the methodcomprising administering a compound that inhibits VEGF-B signaling. Inone example, the blood-brain-barrier breakdown or leakage is associatedwith edema. In one example, the blood-brain-barrier breakdown or leakageis caused by an insult, e.g., trauma and/or by ischemia.

In one example of any method described herein, the compound thatinhibits VEGF-B signaling is administered before or after the stroke.For example, the compound is administered prophylactically ortherapeutically. In one example, the compound is administered before thestroke. In one example, the compound is administered after the stroke.

In one example of any method described herein, the compound isadministered before the stroke and is administered to a subject at riskof having a stroke.

An exemplary subject at risk of having a stroke suffers from diabetesand/or obesity. For example, the diabetes is type 2 diabetes.

Additional or alternative characteristics of a subject at risk ofsuffering from a stroke include one or more of the followingcharacteristics:

-   -   has already suffered from a stroke and/or a transient ischemic        attack;    -   has a family history of stroke;    -   suffers from heart disease;    -   has high blood pressure;    -   has high plasma low density lipoprotein levels;    -   has metabolic syndrome    -   has a cardiac abnormality; and/or    -   has undergone surgery.

In one example, the subject is additionally aged 55 years or more, e.g.,65 years or more or 75 years or more.

In one example of any method described herein, the compound thatinhibits VEGF-B signaling is administered after the stroke and themethod comprises administering a combination of the compound thatinhibits VEGF-B signaling and a thrombolytic compound. Accordingly, thecompound that inhibits VEGF-B signaling is administered therapeutically.

In one example of any method described herein, the compound thatinhibits VEGF-B signaling is administered between about 1 and 10 hoursafter onset of stroke symptoms. For example, the compound that inhibitsVEGF-B signaling is administered between about 1 and 5 hours after onsetof stroke symptoms. the compound that inhibits VEGF-B signaling isadministered between about 1 and 4 hours after onset of stroke symptoms.the compound that inhibits VEGF-B signaling is administered about 1 hourafter onset of stroke symptoms.

Symptoms of stroke will be apparent to the skilled person and include,for example, one or more of facial weakness, arm weakness and/ordifficulty with speech. In one example of any method described herein,the compound that inhibits VEGF-B signaling is administered before thethrombolytic compound. For example, the compound that inhibits VEGF-Bsignaling is administered between about 1 hour and about 10 hours beforethe thrombolytic compound. For example, the compound that inhibitsVEGF-B signaling is administered between about 1 hour and about 6 hoursbefore the thrombolytic compound. For example, the compound thatinhibits VEGF-B signaling is administered between about 2 hours andabout 6 hours before the thrombolytic compound. For example, thecompound that inhibits VEGF-B signaling is administered between about 3hours and about 5 hours before the thrombolytic compound. For example,the compound that inhibits VEGF-B signaling is administered about 4hours before the thrombolytic compound.

In one example of any method described herein, the compound thatinhibits VEGF-B signaling is administered about 1 hour after onset ofstroke symptoms and about 4 hours before the thrombolytic compound.

In one example of any method described herein, administration of thecompound that inhibits VEGF-B signaling extends the time in which thethrombolytic compound can safely be administered to the subject. Thus,the present disclosure also provides a method for extending the time inwhich the thrombolytic compound can safely be administered to thesubject, the method comprising administering the compound that inhibitsVEGF-B signaling and then administering the thrombolytic compound.

In one example of any method described herein, the thrombolytic compoundis administered more than two hours after onset of symptoms of a stroke.For example, the thrombolytic compound is administered more than threehours after onset of symptoms of a stroke. For example, the thrombolyticcompound is administered more than four hours after onset of symptoms ofa stroke. For example, the thrombolytic compound is administered morethan five hours after onset of symptoms of a stroke.

In one example of any method described herein, the thrombolytic compoundis administered between about commencement of a symptom of a stroke andabout ten hours after onset of symptoms of a stroke. For example, thethrombolytic compound is administered between about four and about tenhours after onset of symptoms of a stroke. For example, the thrombolyticcompound is administered between about four and about eight hours afteronset of symptoms of a stroke. For example, the thrombolytic compound isadministered between about three and six hours after onset of symptomsof a stroke. For example, the thrombolytic compound is administeredbetween about four and six hours after onset of symptoms of a stroke.For example, the thrombolytic compound is administered about five hoursafter onset of symptoms of a stroke.

In one example of any method described herein, the compound thatinhibits VEGF-B signaling is administered about 1 hour after onset ofstroke symptoms and the thrombolytic compound is administered about 5hours after onset of stroke symptoms.

In one example of any method described herein, the thrombolytic compoundis selected from the group consisting of a tissue plasminogen activator,lanetoplase, reteplase, staphylokinase, streptokinase, anistreplase,desmoteplase or an urokinase.

In one example of any method described herein, the subject has elevatedfasting blood glucose levels. For example, the subject has fasting bloodglucose levels greater than 150 mg/dL. For example, the subject hasfasting blood glucose levels greater than 180 mg/dL. For example, thesubject has fasting blood glucose levels greater than 200 mg/dL. Forexample, the subject has fasting blood glucose levels greater than 300mg/dL. For example, the subject has fasting blood glucose levels greaterthan 400 mg/dL.

In one example, the symptom of stroke is selected from:

-   -   infarct size;    -   reducing incidence of a haemorrhage in a subject;    -   reducing the likelihood of lethal haemorrhage in a subject;        and/or    -   hemorrhage score as assessed by intracerebral hemorrhage score        in the subject; and/or    -   blood-brain-barrier breakdown or leakage in the subject        following stroke.

In one example of any method described herein, the blood-brain-barrierbreakdown or leakage in the subject following stroke may result in brainedema.

Methods for assessing each of the foregoing are known in the art and/ordescribed herein.

Additional effects of stroke are known in the art and/or describedherein and include, for example, a movement disorder such as paralysis,partial paralysis, slurred speech, uncoordinated movement, muscleweakness, hypotonicity, hypertonicity or involuntary abnormal movement.

In one example of any method described herein, the compound(s) is(are)administered in an amount sufficient to have one or more of thefollowing effects:

-   -   reducing infarct size in the subject;    -   reducing hemorrhage score as assessed by intracerebral        hemorrhage score in the subject;    -   reducing blood-brain-barrier breakdown or leakage in the        subject; and/or    -   reducing brain edema in the subject following stroke.

In one example of any method described herein, the stroke is an ischemicstroke. For example, the stroke is a cerebral ischemic stroke.

In one example of any method described herein, a compound that inhibitsVEGF-B signaling binds to VEGF-B.

For example, the compound is a protein comprising an antibody variableregion that binds to or specifically binds to VEGF-B and neutralizesVEGF-B signaling.

In one example of any method described herein, the compound is anantibody mimetic. For example, the compound is a protein comprising anantigen binding domain of an immunoglobulin, e.g., an IgNAR, a camelidantibody or a T cell receptor.

In one example of any method described herein, a compound is a domainantibody (e.g., comprising only a heavy chain variable region or only alight chain variable region that binds to VEGF-B) or a heavy chain onlyantibody (e.g., a camelid antibody or an IgNAR) or variable regionthereof.

In one example of any method described herein, a compound is a proteincomprising a Fv. For example, the protein is selected from the groupconsisting of:

-   (i) a single chain Fv fragment (scFv);-   (ii) a dimeric scFv (di-scFv); or-   (iv) a diabody;-   (v) a triabody;-   (vi) a tetrabody;-   (vii) a Fab;-   (viii) a F(ab′)₂;-   (ix) a Fv; or-   (x) one of (i) to (ix) linked to a constant region of an antibody,    Fc or a heavy chain constant domain (C_(H)) 2 and/or C_(H)3.

In another example of any method described herein, a compound is anantibody. Exemplary antibodies are full-length and/or naked antibodies.

In one example of any method described herein, the compound is a proteinthat is recombinant, chimeric, CDR grafted, humanized, synhumanized,primatized, deimmunized or human.

In one example of any method described herein, the compound is a proteincomprising an antibody variable region that competitively inhibits thebinding of antibody 2H10 to VEGF-B. In one example, the proteincomprises a heavy chain variable region (V_(H)) comprising a sequenceset forth in SEQ ID NO: 3 and a light chain variable region (V_(L))comprising a sequence set forth in SEQ ID NO: 4.

In one example, the compound is a protein comprising a humanizedvariable region of antibody 2H10. For example, the protein comprises avariable region comprising the complementarity determining regions(CDRs) of the V_(H) and/or the V_(L) of antibody 2H10. For example, theprotein comprises:

-   (i) a V_(H) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 3;    -   (b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 3; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 98-108        of SEQ ID NO: 3; and/or-   (ii) a V_(L) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 23-33        of SEQ ID NO: 4;    -   (b) a CDR2 comprising a sequence set forth in amino acids 49-55        of SEQ ID NO: 4; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 88-96        of SEQ ID NO: 4.

In one example of any method described herein, the compound is a proteincomprising a V_(H) and a V_(L), the V_(H) and V_(L) being humanizedvariable regions of antibody 2H10. For example, the protein comprises:

-   (i) a V_(H) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 3;    -   (b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 3; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 98-108        of SEQ ID NO: 3; and-   (ii) a V_(L) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 23-33        of SEQ ID NO: 4;    -   (b) a CDR2 comprising a sequence set forth in amino acids 49-55        of SEQ ID NO: 4; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 88-96        of SEQ ID NO: 4.

As will be apparent to the skilled artisan, a sequence encoded by anucleic acid includes all variants of that sequence that may be producedduring expression.

In one example of any method described herein, the variable region orV_(H) in any of the foregoing paragraphs comprises a sequence set forthin SEQ ID NO: 5.

In one example of any method described herein, the variable region orV_(L) in any of the foregoing paragraphs comprises a sequence set forthin SEQ ID NO: 6.

In one example of any method described herein, the compound is anantibody.

In one example of any method described herein, the compound is anantibody comprising a V_(H) comprising a sequence set forth in SEQ IDNO: 5 and a V_(L) comprising a sequence set forth in SEQ ID NO: 6.

In one example, the protein or antibody is any form of the protein orantibody encoded by a nucleic acid encoding any of the foregoingproteins or antibodies.

In one example, the protein or antibody comprises:

-   (i) a V_(H) comprising:    -   (a) a CDR1 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 12 or comprising an amino acid sequence of        SEQ ID NO: 18;    -   (b) a CDR2 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 13 or comprising an amino acid sequence of        SEQ ID NO: 19; and    -   (c) a CDR3 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 14 or comprising an amino acid sequence of        SEQ ID NO: 20; and/or-   (ii) a V_(L) comprising:    -   (a) a CDR1 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 15 or comprising an amino acid sequence of        SEQ ID NO: 21;    -   (b) a CDR2 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 16 or comprising an amino acid sequence of        SEQ ID NO: 22; and    -   (c) a CDR3 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 17 or comprising an amino acid sequence of        SEQ ID NO: 23.

In one example, the protein or antibody comprises:

-   (i) a V_(H) comprising:    -   (a) a CDR1 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 24 or comprising an amino acid sequence of        SEQ ID NO: 30;    -   (b) a CDR2 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 25 or comprising an amino acid sequence of        SEQ ID NO: 31; and    -   (c) a CDR3 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 26 or comprising an amino acid sequence of        SEQ ID NO: 32; and/or-   (ii) a V_(L) comprising:    -   (a) a CDR1 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 27 or comprising an amino acid sequence of        SEQ ID NO: 33    -   (b) a CDR2 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 28 or comprising an amino acid sequence of        SEQ ID NO: 34; and    -   (c) a CDR3 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 29 or comprising an amino acid sequence of        SEQ ID NO: 35.

In one example, the protein or antibody comprises:

-   (i) a V_(H) comprising:    -   (a) a CDR1 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 36 or comprising an amino acid sequence of        SEQ ID NO: 42;    -   (b) a CDR2 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 37 or comprising an amino acid sequence of        SEQ ID NO: 43; and    -   (c) a CDR3 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 38 or comprising an amino acid sequence of        SEQ ID NO: 44; and/or-   (ii) a V_(L) comprising:    -   (a) a CDR1 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 39 or comprising an amino acid sequence of        SEQ ID NO: 45;    -   (b) a CDR2 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 40 or comprising an amino acid sequence of        SEQ ID NO: 46; and    -   (c) a CDR3 comprising a sequence encoded by a nucleic acid        comprising SEQ ID NO: 41 or comprising an amino acid sequence of        SEQ ID NO: 47.

In one example, the compound is within a composition. For example, thecomposition comprises a protein comprising an antibody variable regionor a V_(H) or a V_(L) or an antibody as described herein. In oneexample, the composition additionally comprises one or more variants ofthe protein or antibody. For example, that comprises a variant missingan encoded C-terminal lysine residue, a deamidated variant and/or aglycosylated variant and/or a variant comprising a pyroglutamate, e.g.,at the N-terminus of a protein and/or a variant lacking a N-terminalresidue, e.g., a N-terminal glutamine in an antibody or V region and/ora variant comprising all or part of a secretion signal. Deamidatedvariants of encoded asparigine residues may result in isoaspartic, andaspartic acid isoforms being generated or even a succinamide involvingan adjacent amino acid residue. Deamidated variants of encoded glutamineresidues may result in glutamic acid. Compositions comprising aheterogeneous mixture of such sequences and variants are intended to beincluded when reference is made to a particular amino acid sequence.

In one example of any method described herein, the compound thatinhibits VEGF-B signaling inhibits or prevents expression of VEGF-B. Forexample, the compound is selected from the group an antisense, a siRNA,a RNAi, a ribozyme and a DNAzyme.

In one example of any method described herein, the VEGF-B is mammalianVEGF-B, e.g., human VEGF-B.

In one example of any method described herein, the subject is a mammal,for example a primate, such as a human.

Methods of treatment described herein can additionally compriseadministering a further compound to reduce, treat or prevent the effectof a stroke.

The present disclosure also provides a compound that inhibits VEGF-Bsignaling for use in reducing an effect of stroke.

The present disclosure also provides for use of a compound that inhibitsVEGF-B signaling in the manufacture of a medicament for reducing aneffect of stroke.

The present disclosure also provides a kit comprising a compound thatinhibits VEGF-B signaling packaged with instructions for use in reducingan effect of stroke. Optionally, the kit additionally comprises athrombolytic compound.

The present disclosure also provides a kit comprising a compound thatinhibits VEGF-B signaling packaged with instructions to administer thecompound to a subject who has suffered a stroke in combination with athrombolytic compound.

The present disclosure also provides a kit comprising a thrombolyticcompound packaged with instructions to administer the compound to asubject who has suffered a stroke in combination with a compound thatinhibits VEGF-B signaling.

Exemplary effects of stroke and compounds are described herein and areto be taken to apply mutatis mutandis to the examples of the disclosureset out in the previous six paragraphs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graphical representation showing endothelial cell uptake offatty acids is significantly increased after 2 hours treatment withVEGF-B proteins as measured using the BODIPY-C12 tracer. Values areratio of uptake normalized to control. *P<0.05, ***P<0.001 compared tocontrol.

FIG. 1B is a graphical representation showing endothelial cell glucoseuptake is significantly decreased after 2 hours treatment with VEGF-Bproteins as measured using a fluorescent glucose analogue (2-NBDG).Values are ratio of uptake normalized to control. *P<0.05, **P<0.01,***P<0.001 compared to control.

FIG. 1C is a graphical representation showing primary endothelial cellsexposed to fatty acids (FA; 25 μM sodium palmitate and 25 μM sodiumoleate) for 2 hours or overnight (o/n) show decreased ability to take upglucose (as measured using 2-NBDG). Values are means±SEM. **P<0.01compared to controls.

FIG. 2A is a graphical representation showing elevated blood glucoselevels in diet-induced obese (DIO) mice compared with age-matched miceon normal chow. Values are means±SEM. ***P<0.001 compared to controls.

FIG. 2B is a graphical representation showing larger infarct volumes inDIO mice compared with age-matched mice on normal chow followingischemic stroke. Infarct volumes were measured at 72 h by TTC staining.Values are means±SEM, n=10 per group. *P<0.01 compared to controls.

FIG. 2C is a graphical representation showing increased hemorrhage scorein DIO mice compared with age-matched mice on normal chow followingischemic stroke. Hemorrhage was assessed at 72 h by a scoring system.Values are means ±SEM, n=10 per group. *P<0.01 compared to controls.

FIG. 2D is a graphical representation showing Nrp1 expression asmeasured by qPCR is increased in DIO mice 3 hours post ischemic stroke,whilst transcript levels of Vegfb and its receptor Vegfr1 (Flt1) werenot significantly different to contralateral controls. Values aremeans±SEM, n=3 per group. **P<0.01 compared to controls.

FIG. 3A is a graphical representation showing VEGF-B antagonism with2H10 improves glucose analogue ¹⁸F-deoxyglucose (18FDG) uptake in thebrains of DIO mice compared to mice treated with isotype control IgG.Glucose analogue uptake in 2H10 treated diet-induced obese mice wascomparable to that observed in lean mice. n=2-5/group, Mean±SEM.#p<0.05, **p<0.01.

FIG. 3B is a graphical representation showing pre-treatment with theVEGF-B blocking antibody 2H10 decreases infarct volumes in DIO micecompared with age-matched mice on normal chow following ischemic stroke.Infarct volumes were measured at 72 h by2,3,5-triphenyltetrazolium-chloride (TTC) staining. Values aremeans±SEM, n=10 per group. **P<0.01 compared to controls.

FIG. 3C is a graphical representation showing pre-treatment with theVEGF-B blocking antibody 2H10 decreases hemorrhage score in DIO micecompared with age-matched mice on normal chow following ischemic stroke.Hemorrhage was assessed at 72 h by a scoring system. Values aremeans±SEM, n=10 per group. **P<0.01 compared to controls.

FIG. 3D is a graphical representation showing retained expression of theglucose transporter Glut-1 in the ischemic penumbra 1 hour afterischemic stroke following prophylactic treatment with the anti-VEGF-Bantibody 2H10. N=5/group. Mean±SEM, *p<0.05 vs contra, #p<0.05 vscontrol.

FIG. 3E is a graphical representation showing prophylactic treatmentwith the anti-VEGF-B antibody 2H10 in DIO mice reducesblood-brain-barrier leakage (as measured by Dextran extravasation) 1hour after ischemic stroke. N=5/group, mean±SEM, ** p<0.01.

FIG. 3F is a graphical representation showing prophylactic anti-VEGF-Bantibody treatment decreases Occludin serine phosphorylation (serineresidue 490) following ischemic stroke in DIO mice compared to IgGcontrol treated mice. Staining with a phosphoserine specificanti-Occludin antibody in the contralateral hemisphere and ipsilateralpenumbra region were quantified and expressed as pixel intensity. N=3,*p<0.05.

FIG. 3G is a graphical representation showing lipid accumulation (asmeasured by vascular adipophilin immunostaining) in cerebral bloodvessels 3 h post ischemic stroke in DIO mice is blocked by prophylactictreatment with the anti-VEGF-B antibody 2H10 compared to isotype controltreatment (IgG). Mean±SEM, **p<0.01.

FIG. 4A is a graphical representation showing inhibition of VEGF-B with2H10 antibody significantly reduces stroke volume after latethrombolysis in DIO mice following ischemic stroke. Infarct volumes weremeasure 72 hours after ischemic stroke by TTC staining. N=6-10/group,mean+SEM, **p<0.01.

FIG. 4B is a graphical representation showing inhibition of VEGF-B with2H10 antibody significantly reduces hemorrhage score after latethrombolysis in DIO mice following ischemic stroke. Hemorrhage score wasmeasured histologically 72 hours after ischemic stroke. N=6-10/group,mean+SEM, **p<0.01.

FIG. 4C is a Kaplan Meier survival analysis showing inhibition of VEGF-Bwith 2H10 antibodies improves survival outcomes after late thrombolysisin mice with diet-induced obesity following ischemic stroke. N=10/group,mean+SEM, *p<0.05.

KEY TO SEQUENCE LISTING

-   SEQ ID NO: 1 is an amino acid sequence of a human VEGF-B₁₈₆ isoform    containing a 21 amino acid N-terminal signal sequence-   SEQ ID NO: 2 is an amino acid sequence of a human VEGF-B₁₆₇ isoform    containing a 21 amino acid N-terminal signal sequence-   SEQ ID NO: 3 is an amino acid sequence from a V_(H) of antibody    2H10.-   SEQ ID NO: 4 is an amino acid sequence from a V_(L) of antibody    2H10.-   SEQ ID NO: 5 is an amino acid sequence from a V_(H) of a humanized    form of antibody 2H10.-   SEQ ID NO: 6 is an amino acid sequence of a V_(L) of a humanized    form of antibody 2H10.-   SEQ ID NO: 7 is an amino acid sequence from a V_(H) of antibody    4E12.-   SEQ ID NO: 8 is an amino acid sequence of a V_(L) of antibody 4E12.-   SEQ ID NO: 9 is an amino acid sequence from a V_(H) of antibody 2F5.-   SEQ ID NO: 10 is an amino acid sequence of a V_(L) of antibody 2F5.-   SEQ ID NO: 11 is an amino acid sequence of recombinant human tissue    plasminogen activator.-   SEQ ID NO: 12 is a nucleotide sequence from a V_(L) CDR1 of antibody    2H10-   SEQ ID NO: 13 is a nucleotide sequence from a V_(L) CDR2 of antibody    2H10-   SEQ ID NO: 14 is a nucleotide sequence from a V_(L) CDR3 of antibody    2H10-   SEQ ID NO: 15 is a nucleotide sequence from a V_(H) CDR1 of antibody    2H10-   SEQ ID NO: 16 is a nucleotide sequence from a V_(H) CDR2 of antibody    2H10-   SEQ ID NO: 17 is a nucleotide sequence from a V_(H) CDR3 of antibody    2H10-   SEQ ID NO: 18 is an amino acid sequence from a V_(L) CDR1 of    antibody 2H10-   SEQ ID NO: 19 is an amino acid sequence from a V_(L) CDR2 of    antibody 2H10-   SEQ ID NO: 20 is an amino acid sequence from a V_(L) CDR3 of    antibody 2H10-   SEQ ID NO: 21 is an amino acid sequence from a V_(H) CDR1 of    antibody 2H10-   SEQ ID NO: 22 is an amino acid sequence from a V_(H) CDR2 of    antibody 2H10-   SEQ ID NO: 23 is an amino acid sequence from a V_(H) CDR3 of    antibody 2H10-   SEQ ID NO: 24 is a nucleotide sequence from a V_(L) CDR1 of antibody    2F5-   SEQ ID NO: 25 is a nucleotide sequence from a V_(L) CDR2 of antibody    2F5-   SEQ ID NO: 26 is a nucleotide sequence from a V_(L) CDR3 of antibody    2F5-   SEQ ID NO: 27 is a nucleotide sequence from a V_(H) CDR1 of antibody    2F5-   SEQ ID NO: 28 is a nucleotide sequence from a V_(H) CDR2 of antibody    2F5-   SEQ ID NO: 29 is a nucleotide sequence from a V_(H) CDR3 of antibody    2F5-   SEQ ID NO: 30 is an amino acid sequence from a V_(L) CDR1 of    antibody 2F5-   SEQ ID NO: 31 is an amino acid sequence from a V_(L) CDR2 of    antibody 2F5-   SEQ ID NO: 32 is an amino acid sequence from a V_(L) CDR3 of    antibody 2F5-   SEQ ID NO: 33 is an amino acid sequence from a V_(H) CDR1 of    antibody 2F5-   SEQ ID NO: 34 is an amino acid sequence from a V_(H) CDR2 of    antibody 2F5-   SEQ ID NO: 35 is an amino acid sequence from a V_(H) CDR3 of    antibody 2F5-   SEQ ID NO: 36 is a nucleotide sequence from a V_(L) CDR1 of antibody    4E12-   SEQ ID NO: 37 is a nucleotide sequence from a V_(L) CDR2 of antibody    4E12-   SEQ ID NO: 38 is a nucleotide sequence from a V_(L) CDR3 of antibody    4E12-   SEQ ID NO: 39 is a nucleotide sequence from a V_(H) CDR1 of antibody    4E12-   SEQ ID NO: 40 is a nucleotide sequence from a V_(H) CDR2 of antibody    4E12-   SEQ ID NO: 41 is a nucleotide sequence from a V_(H) CDR3 of antibody    4E12-   SEQ ID NO: 42 is an amino acid sequence from a V_(L) CDR1 of    antibody 4E12-   SEQ ID NO: 43 is an amino acid sequence from a V_(L) CDR2 of    antibody 4E12-   SEQ ID NO: 44 is an amino acid sequence from a V_(L) CDR3 of    antibody 4E12-   SEQ ID NO: 45 is an amino acid sequence from a V_(H) CDR1 of    antibody 4E12-   SEQ ID NO: 46 is an amino acid sequence from a V_(H) CDR2 of    antibody 4E12-   SEQ ID NO: 47 is an amino acid sequence from a V_(H) CDR3 of    antibody 4E12

DESCRIPTION General

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or groups of compositionsof matter.

Those skilled in the art will appreciate that the present disclosure issusceptible to variations and modifications other than thosespecifically described. It is to be understood that the disclosureincludes all such variations and modifications. The disclosure alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specificexamples described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to applymutatis mutandis to any other example of the disclosure unlessspecifically stated otherwise.

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (for example, in cellculture, molecular genetics, immunology, immunohistochemistry, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present disclosure are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The description and definitions of variable regions and parts thereof,immunoglobulins, antibodies and fragments thereof herein may be furtherclarified by the discussion in Kabat Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia andLesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883,1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

Any discussion of a protein or antibody herein will be understood toinclude any variants of the protein or antibody produced duringmanufacturing and/or storage. For example, during manufacturing orstorage an antibody can be deamidated (e.g., at an asparagine or aglutamine residue) and/or have altered glycosylation and/or have aglutamine residue converted to pyroglutamate and/or have a N-terminal orC-terminal residue removed or “clipped” and/or have part or all of asignal sequence incompletely processed and, as a consequence, remain atthe terminus of the antibody. It is understood that a compositioncomprising a particular amino acid sequence may be a heterogeneousmixture of the stated or encoded sequence and/or variants of that statedor encoded sequence.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source.

Selected Definitions

VEGF-B is known to exist in two major isoforms, referred to as VEGF-B₁₈₆and VEGF-B₁₆₇. For the purposes of nomenclature only and not limitationexemplary sequences of human VEGF-B₁₈₆ is set out in NCBI ReferenceSequence: NP_003368.1, in NCBI protein accession numbers NP_003368,P49765 and AAL79001 and in SEQ ID NO: 1. In the context of the presentdisclosure, the sequence of VEGF-B₁₈₆ can lack the 21 amino acidN-terminal signal sequence (e.g., as set out at amino acids 1 to 21 ofSEQ ID NO: 1. For the purposes of nomenclature only and not limitationexemplary sequences of human VEGF-B₁₆₇ is set out in NCBI ReferenceSequence: NP_001230662.1, in NCBI protein accession numbers AAL79000 andAAB06274 and in SEQ ID NO: 2. In the context of the present disclosure,the sequence of VEGF-B₁₆₇ can lack the 21 amino acid N-terminal signalsequence (e.g., as set out at amino acids 1 to 21 of SEQ ID NO: 2.Additional sequence of VEGF-B can be determined using sequences providedherein and/or in publically available databases and/or determined usingstandard techniques (e.g., as described in Ausubel et al., (editors),Current Protocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present) orSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (1989)). Reference to human VEGF-B may beabbreviated to hVEGF-B. In one example, reference herein to VEGF-B is toVEGF-B₁₆₇ isoform.

Reference herein to VEGF-B also encompasses the VEGF-B₁₀₋₁₀₈ peptide asdescribed in WO2006/012688.

As used herein, the term “stroke” shall be taken to mean loss of brainfunction(s), usually rapidly developing, that is due to a disturbance inblood flow to the brain or brainstem. The disturbance can be ischemia(lack of blood) caused by, e.g., thrombosis or embolism, or can be dueto a hemorrhage. In one example, the loss of brain function isaccompanied by neuronal cell death. In one example, the stroke is causedby a disturbance or loss of blood from the cerebrum or a region thereof.In one example, a stroke is a neurological deficit of cerebrovascularcause that persists beyond 24 hours or is interrupted by death within 24hours (as defined by the World Health Organization). Persistence ofsymptoms beyond 24 hours separates stroke from Transient Ischemic Attack(TIA), in which symptoms persist for less than 24 hours. Symptoms ofstroke include hemiplegia (paralysis of one side of the body);hemiparesis (weakness on one side of the body); muscle weakness of theface; numbness; reduction in sensation; altered sense of smell, sense oftaste, hearing, or vision; loss of smell, taste, hearing, or vision;drooping of an eyelid (ptosis); detectable weakness of an ocular muscle;decreased gag reflex; decreased ability to swallow; decreased pupilreactivity to light; decreased sensation of the face; decreased balance;nystagmus; altered breathing rate; altered heart rate; weakness insternocleidomastoid muscle with decreased ability or inability to turnthe head to one side; weakness in the tongue; aphasia (inability tospeak or understand language); apraxia (altered voluntary movements); avisual field defect; a memory deficit; hemineglect or hemispatialneglect (deficit in attention to the space on the side of the visualfield opposite the lesion); disorganized thinking; confusion;development of hypersexual gestures; anosognosia (persistent denial ofthe existence of a deficit); difficulty walking; altered movementcoordination; vertigo; disequilibrium; loss of consciousness; headache;and/or vomiting.

The term “effect of stroke” will be understood to include one or more ofinfarct size, hemorrhage score and/or blood brain barrier breakdown orleakage. However, this term is not to be limited to these effects andencompasses any change in a subject, such as any clinical change in asubject e.g., neurological or physical change, that results from astroke. Such changes or effects include a movement disorder, loss ofcerebral function or any of the symptoms described herein.

The term “onset of stroke symptoms” will be understood to refer to atime at which a subject or another person recognizes one or moresymptoms of stroke. Suitable symptoms are described herein.

As used herein, the term “incidence of hemorrhage” will be understood tomean the number of or size of hemorrhages suffered by a subject or apopulation of subjects. Thus, a reduction in the incidence of hemorrhagein a subject can be a reduction in the number or size of hemorrhages inthe subject or a reduction in the likelihood that a subject will sufferfrom one or more hemorrhages as a result of a stroke.

The term “likelihood of lethal hemorrhage” will be understood to meanthat a subject to which the compound(s) has been administered is lesslikely to die as a result of a hemorrhage than a subject who has alsosuffered from a stroke and who has not been administered thecompound(s). Clearly such likelihoods can be calculated on the basis ofpopulation data rather than requiring a side-by-side comparison for eachsubject. This term will also provide explicit support for reducing thelikelihood of death as a result of stroke, i.e., the disclosureadditionally provides methods of reducing the likelihood of death as aresult of stroke. All method steps described above in relation toreducing an effect of stroke will be taken to apply equally to suchmethods.

The term “blood brain barrier” shall me taken to mean the highlyselective permeability barrier that separates the circulating blood fromthe brain extra-cellular fluid in the central nervous system. Breakdownor leakage of the blood brain barrier may result in accumulation offluid in the brain's extra-cellular space or cerebral edema. Breakdownof the blood brain barrier may result from a traumatic brain injury orfrom non-traumatic causes such as ischemic stroke, cancer, or braininflammation due to meningitis or encephalitis.

The term “thrombolytic compound” shall be taken to mean a compound thatinduces or mediates or enhances breakdown of one or more blood clots tolimit occlusion of blood vessels. The thrombolytic compound can act bystimulating secondary fibrinolysis by plasmin. Exemplary thrombolyticagents include tissue plasminogen activator (tPA), anistreplase (APSAC),streptokinase (SK), staphylokinase (SAK), desmoteplase or urokinase(uPA).

In one example, the thrombolytic compound is “tissue plasminogenactivator” or tPA. tPA is a serine protease that is involved in thebreakdown of blood clots by catalyzing the conversion of plasminogen toplasmin. In one example, the tissue plasminogen activator (tPA) may bemanufactured using recombinant techniques. Accordingly it may berecombinant tissue plasminogen activator (r-tPA), for example comprisinga sequence set forth in SEQ ID NO: 11. Exemplary tPAs include alteplase,reteplase, lanoteplase or tenecteplase.

As used herein, reference to “effective administration” (or similar) ofa thrombolytic compound will mean that the administration of thethrombolytic compound provides a clinically effective outcome, i.e., animprovement in reducing the effect of a stroke compared to that seen ina subject or population of subjects in which the same thombolyticcompound is administered in the same manner (e.g., at the same timeand/or patients having similar blood glucose levels) with beingadministered an inhibitor of VEGF-B signaling.

As will be apparent to the skilled artisan based on the descriptionherein, when discussing administering a “combination” of compounds, thepresent disclosure contemplates administering the compounds as a singlecomposition, administering the compounds at the same time (but asseparate compositions) or administering the compounds sequentially.

The term “recombinant” shall be understood to mean the product ofartificial genetic recombination. Accordingly, in the context of arecombinant protein comprising an antibody variable region, this termdoes not encompass an antibody naturally-occurring within a subject'sbody that is the product of natural recombination that occurs during Bcell maturation. However, if such an antibody is isolated, it is to beconsidered an isolated protein comprising an antibody variable region.Similarly, if nucleic acid encoding the protein is isolated andexpressed using recombinant means, the resulting protein is arecombinant protein comprising an antibody variable region. Arecombinant protein also encompasses a protein expressed by artificialrecombinant means when it is within a cell, tissue or subject, e.g., inwhich it is expressed.

The term “protein” shall be taken to include a single polypeptide chain,i.e., a series of contiguous amino acids linked by peptide bonds or aseries of polypeptide chains covalently or non-covalently linked to oneanother (i.e., a polypeptide complex). For example, the series ofpolypeptide chains can be covalently linked using a suitable chemical ora disulfide bond. Examples of non-covalent bonds include hydrogen bonds,ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term “polypeptide” or “polypeptide chain” will be understood fromthe foregoing paragraph to mean a series of contiguous amino acidslinked by peptide bonds.

The skilled artisan will be aware that an “antibody” is generallyconsidered to be a protein that comprises a variable region made up of aplurality of polypeptide chains, e.g., a polypeptide comprising a lightchain variable region (V_(L)) and a polypeptide comprising a heavy chainvariable region (V_(H)). An antibody also generally comprises constantdomains, some of which can be arranged into a constant region, whichincludes a constant fragment or fragment crystallizable (Fc), in thecase of a heavy chain. A V_(H) and a V_(L) interact to form a Fvcomprising an antigen binding region that is capable of specificallybinding to one or a few closely related antigens. Generally, a lightchain from mammals is either a κ light chain or a λ light chain and aheavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁ and IgA₂) or subclass. The term “antibody” alsoencompasses humanized antibodies, primatized antibodies, humanantibodies, synhumanized antibodies and chimeric antibodies.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antigen binding fragment of an antibody.Specifically, whole antibodies include those with heavy and light chainsincluding an Fc region. The constant domains may be wild-type sequenceconstant domains (e.g., human wild-type sequence constant domains) oramino acid sequence variants thereof.

As used herein, “variable region” refers to the portions of the lightand/or heavy chains of an antibody as defined herein that is capable ofspecifically binding to an antigen and includes amino acid sequences ofcomplementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3,and framework regions (FRs). Exemplary variable regions comprise threeor four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with threeCDRs. In the case of a protein derived from an IgNAR, the protein maylack a CDR2. V_(H) refers to the variable region of the heavy chain.V_(L) refers to the variable region of the light chain.

As used herein, the term “complementarity determining regions” (syn.CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues ofan antibody variable domain the presence of which are necessary forantigen binding. Each variable domain typically has three CDR regionsidentified as CDR1, CDR2 and CDR3. The amino acid positions assigned toCDRs and FRs can be defined according to Kabat Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,1987 and 1991 or other numbering systems in the performance of thisdisclosure, e.g., the canonical numbering system of Chothia and Lesk J.Mol Biol. 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989;and/or Al-Lazikani et al., J Mol Biol 273: 927-948, 1997; the IMGTnumbering system of Lefranc et al., Devel. And Compar. Immunol., 27:55-77, 2003; or the AHO numbering system of Honnegher and Plükthun J.Mol. Biol., 309: 657-670, 2001.

“Framework regions” (FRs) are those variable domain residues other thanthe CDR residues.

As used herein, the term “Fv” shall be taken to mean any protein,whether comprised of multiple polypeptides or a single polypeptide, inwhich a V_(L) and a V_(H) associate and form a complex having an antigenbinding site, i.e., capable of specifically binding to an antigen. TheV_(H) and the V_(L) which form the antigen binding site can be in asingle polypeptide chain or in different polypeptide chains.Furthermore, an Fv of the disclosure (as well as any protein of thedisclosure) may have multiple antigen binding sites which may or may notbind the same antigen. This term shall be understood to encompassfragments directly derived from an antibody as well as proteinscorresponding to such a fragment produced using recombinant means. Insome examples, the V_(H) is not linked to a heavy chain constant domain(C_(H)) 1 and/or the V_(L) is not linked to a light chain constantdomain (C_(L)). Exemplary Fv containing polypeptides or proteins includea Fab fragment, a Fab′ fragment, a F(ab′) fragment, a scFv, a diabody, atriabody, a tetrabody or higher order complex, or any of the foregoinglinked to a constant region or domain thereof, e.g., C_(H)2 or C_(H)3domain, e.g., a minibody. A “Fab fragment” consists of a monovalentantigen-binding fragment of an antibody, and can be produced bydigestion of a whole antibody with the enzyme papain, to yield afragment consisting of an intact light chain and a portion of a heavychain or can be produced using recombinant means. A “Fab′ fragment” ofan antibody can be obtained by treating a whole antibody with pepsin,followed by reduction, to yield a molecule consisting of an intact lightchain and a portion of a heavy chain comprising a V_(H) and a singleconstant domain. Two Fab′ fragments are obtained per antibody treated inthis manner. A Fab′ fragment can also be produced by recombinant means.A “F(ab′)2 fragment” of an antibody consists of a dimer of two Fab′fragments held together by two disulfide bonds, and is obtained bytreating a whole antibody molecule with the enzyme pepsin, withoutsubsequent reduction. A “Fab₂” fragment is a recombinant fragmentcomprising two Fab fragments linked using, for example a leucine zipperor a C_(H)3 domain. A “single chain Fv” or “scFv” is a recombinantmolecule containing the variable region fragment (Fv) of an antibody inwhich the variable region of the light chain and the variable region ofthe heavy chain are covalently linked by a suitable, flexiblepolypeptide linker.

As used herein, the term “binds” in reference to the interaction of aprotein or an antigen binding site thereof with an antigen means thatthe interaction is dependent upon the presence of a particular structure(e.g., an antigenic determinant or epitope) on the antigen. For example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody binds to epitope “A”, thepresence of a molecule containing epitope “A” (or free, unlabeled “A”),in a reaction containing labeled “A” and the protein, will reduce theamount of labeled “A” bound to the antibody.

As used herein, the term “specifically binds” or “binds specifically”shall be taken to mean that a protein of the disclosure reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular antigen or cell expressing samethan it does with alternative antigens or cells. For example, a proteinbinds to VEGF-B with materially greater affinity (e.g., 20 fold or 40fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than itdoes to other growth factor (e.g., VEGF-A) or to antigens commonlyrecognized by polyreactive natural antibodies (i.e., by naturallyoccurring antibodies known to bind a variety of antigens naturally foundin humans). Generally, but not necessarily, reference to binding meansspecific binding, and each term shall be understood to provide explicitsupport for the other term.

As used herein, the term “neutralize” shall be taken to mean that aprotein is capable of blocking, reducing or preventing VEGF-B-signalingin a cell through the VEGF-R1. Methods for determining neutralizationare known in the art and/or described herein.

As used herein, the terms “preventing”, “prevent” or “prevention”include administering a compound of the disclosure to thereby stop orhinder the development of at least one symptom of a condition.

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a protein described herein to thereby reduce or eliminateat least one symptom of a specified disease or condition or to slowprogression of the disease or condition.

As used herein, the term “subject” shall be taken to mean any animalincluding humans, for example a mammal. Exemplary subjects include butare not limited to humans and non-human primates. For example, thesubject is a human.

Reducing an Effect of Stroke

The disclosure herein provides, for example, a method that reduces oneor more effects of stroke in a subject comprising administering to thesubject a compound that inhibits VEGF-B signaling.

In one example, the subject suffers from diabetes. For example, asubject suffering from diabetes has a clinically accepted marker ofdiabetes, such as:

-   -   Fasting plasma glucose of greater than or equal to 7 nmol/L or        126 mg/dl;    -   Casual plasma glucose (taken at any time of the day) of greater        than or equal to 11.1 nmol/L or 200 mg/dl with the symptoms of        diabetes.    -   Oral glucose tolerance test (OGTT) value of greater than or        equal to 11.1 nmol/L or 200 mg/dl measured at a two-hour        interval. The OGTT is given over a two or three-hour time span.

In one example, the subject suffers from type 1 diabetes.

In one example, the subject suffers from type 2 diabetes.

The methods of the present disclosure can be readily applied to any formof ischemia in the central nervous system. For example, the subject canpresent with sign(s) and/or symptoms of retinal ischemia. Thus, themethods of the present disclosure will be taken to apply to reducing theeffect of ischemia in the central nervous system, e.g., retinalischemia.

In one example, the subject is at risk of stroke but the onset of stokehas not yet occurred. A subject is at risk if he or she has a higherrisk of developing stroke than a control population. The controlpopulation may include one or more subjects selected at random from thegeneral population (e.g., matched by age, gender, race and/or ethnicity)who have not been diagnosed or have a family history of stroke. Asubject can be considered at risk for a stroke if a “risk factor”associated with that stroke is found to be associated with that subject.A risk factor can include any activity, trait, event or propertyassociated with a given disorder, for example, through statistical orepidemiological studies on a population of subjects. A subject can thusbe classified as being at risk for a stroke even if studies identifyingthe underlying risk factors did not include the subject specifically.For example, a subject undergoing heart surgery is at risk of transientcerebral ischemic attack (or stroke) because the frequency of transientcerebral ischemic attack is increased in a population of subjects whohave undergone heart surgery as compared to a population of subjects whohave not.

In one example, a subject at risk of stroke include those undergoing asurgical procedure on the brain or central nervous system, such asendovascular surgery, clipping, stenting or microcathetherization. Suchsubjects also include those undergoing surgery elsewhere in the bodythat affects a blood vessel supplying the brain (that is connecting thebrain to the heart, for example, carotid arteries and jugular veins) oron an artery supplying blood to the retina. An exemplary class ofsubjects are those undergoing endovascular surgery to treat a brainaneurysm. Subjects undergoing these types of surgery are at enhancedrisk of stroke.

In one example subjects at risk of stroke also include patients who aresmokers, hypertensive, diabetic, hyper-cholesterolemic. Subjectsespecially at a high risk are those who have had a prior stroke, minorstroke, or transient ischemic attack.

As discussed above, methods of the disclosure achieve one or more of thefollowing effects:

-   -   reducing infarct size in the subject;    -   reducing hemorrhage score as assessed by intracerebral        hemorrhage score in the subject; and/or    -   reducing blood-brain-barrier breakdown or leakage in the        subject; and/or    -   reducing cerebral edemal in the subject following stroke.

Methods for assessing infarct size are known in the art and include, forexample, echnetium-99m sestamibi single-photon emission computedtomography (SPECT), computed tomography, or magnetic resonance imaging.

Methods for assessing intracerebral hemorrhage score are described, forexample, in Hemphil et al., Stroke, 32: 891-897, 2001. The presence ofhemorrhage, e.g., intracerebral hemorrhage can be determined using,e.g., MRI or CT scanning.

Blood brain barrier breakdown/leakage/permeability can also be detectedusing MRI, optionally using a tracer.

In one example, a method of the disclosure reduces any symptom of strokeknown in the art or described herein.

As will be apparent to the skilled person a “reduction” in a symptom oreffect of stroke in a subject will be comparative to another subject whohas also suffered a stroke but who has not received treatment with amethod described herein. This does not necessarily require aside-by-side comparison of two subjects. Rather population data can berelied upon. For example a population of subjects suffering from strokewho have not received treatment with a method described herein(optionally, a population of similar subjects to the treated subject,e.g., age, weight, diabetic status, blood glucose levels) are assessedand the mean values are compared to results of a subject or populationof subjects treated with a method described herein.

VEGF-B Signaling Inhibitors Proteins Comprising Antibody VariableRegions

An exemplary VEGF-B signaling inhibitor comprises an antibody variableregion, e.g., is an antibody or an antibody fragment that binds toVEGF-B and neutralizes VEGF-B signaling.

In one example, the antibody variable region binds specifically toVEGF-B.

Suitable antibodies and proteins comprising variable regions thereof areknown in the art.

For example, anti-VEGF-B antibodies and fragments thereof are describedin WO2006/012688.

In one example, the anti-VEGF-B antibody or fragment thereof is anantibody that competitively inhibits the binding of 2H10 to VEGF-B or anantigen binding fragment thereof. In one example, the anti-VEGF-Bantibody or fragment thereof is antibody 2H10 or a chimeric, CDR graftedor humanized version thereof or an antigen binding fragment thereof . Inthis regard, antibody 2H10 comprises a V_(H) comprising a sequence setforth in SEQ ID NO: 3 and a V_(L) comprising a sequence set forth in SEQID NO: 4. Exemplary chimeric and humanized versions of this antibody aredescribed in WO2006/012688.

In one example, the anti-VEGF-B antibody or fragment thereof comprises aV_(H) comprising a sequence set forth in SEQ ID NO: 5 and a V_(L)comprising a sequence set forth in SEQ ID NO: 6.

In one example, the anti-VEGF-B antibody or fragment thereof is anantibody that competitively inhibits the binding of 4E12 to VEGF-B or anantigen binding fragment thereof. In one example, the anti-VEGF-Bantibody or fragment thereof is antibody 4E12 or a chimeric, CDR graftedor humanized version thereof or an antigen binding fragment thereof. Inthis regard, antibody 4E12 comprises a V_(H) comprising a sequence setforth in SEQ ID NO: 7 and a V_(L) comprising a sequence set forth in SEQID NO: 8.

In one example, the compound is a protein comprising a humanizedvariable region of antibody 4E12. For example, the protein comprises avariable region comprising the complementarity determining regions(CDRs) of the V_(H) and/or the V_(L) of antibody 4E12. For example, theprotein comprises:

-   (i) a V_(H) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 7;    -   (b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 7; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 98-105        of SEQ ID NO: 7; and/or-   (ii) a V_(L) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 24-34        of SEQ ID NO: 8;    -   (b) a CDR2 comprising a sequence set forth in amino acids 50-56        of SEQ ID NO: 8; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 89-97        of SEQ ID NO: 8.

In one example, the anti-VEGF-B antibody or fragment thereof is anantibody that competitively inhibits the binding of 2F5 to VEGF-B or anantigen binding fragment thereof. In one example, the anti-VEGF-Bantibody or fragment thereof is antibody 2F5 or a chimeric, CDR graftedor humanized version thereof or an antigen binding fragment thereof. Inthis regard, antibody 2E5 comprises a V_(H) comprising a sequence setforth in SEQ ID NO: 9 and a V_(L) comprising a sequence set forth in SEQID NO: 10.

In one example, the compound is a protein comprising a humanizedvariable region of antibody 2F5. For example, the protein comprises avariable region comprising the complementarity determining regions(CDRs) of the V_(H) and/or the V_(L) of antibody 2F5.

For example, the protein comprises:

-   (i) a V_(H) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 9;    -   (b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 9; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 98-107        of SEQ ID NO: 9; and/or-   (ii) a V_(L) comprising:    -   (a) a CDR1 comprising a sequence set forth in amino acids 24-34        of SEQ ID NO: 10;    -   (b) a CDR2 comprising a sequence set forth in amino acids 50-56        of SEQ ID NO: 10; and    -   (c) a CDR3 comprising a sequence set forth in amino acids 89-96        of SEQ ID NO: 10.

In another example, an antibody or protein comprising a variable regionthereof is produced using a standard method, e.g., as is known in theart or briefly described herein.

Immunization-Based Methods

To generate antibodies, VEGF-B or an epitope bearing fragment or portionthereof or a modified form thereof or nucleic acid encoding same (an“immunogen”), optionally formulated with any suitable or desiredadjuvant and/or pharmaceutically acceptable carrier, is administered toa subject (for example, a non-human animal subject, such as, a mouse, arat, a chicken etc.) in the form of an injectable composition. Exemplarynon-human animals are mammals, such as murine animals (e.g., rats ormice). Injection may be intranasal, intramuscular, sub-cutaneous,intravenous, intradermal, intraperitoneal, or by other known route.Optionally, the immunogen is administered numerous times. Means forpreparing and characterizing antibodies are known in the art (See, e.g.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).Methods for producing anti-VEGF-B antibodies in mice are described inWO2006/012688.

The production of polyclonal antibodies may be monitored by samplingblood of the immunized animal at various points following immunization.A second, booster injection, may be given, if required to achieve adesired antibody titer. The process of boosting and titering is repeateduntil a suitable titer is achieved. When a desired level ofimmunogenicity is obtained, the immunized animal is bled and the serumisolated and stored, and/or the animal is used to generate monoclonalantibodies (mAbs).

Monoclonal antibodies are exemplary antibodies contemplated by thepresent disclosure. Generally, production of monoclonal antibodiesinvolves, immunizing a subject (e.g., a rodent, e.g., mouse or rat) withthe immunogen under conditions sufficient to stimulate antibodyproducing cells. In some examples, a mouse genetically-engineered toexpress human antibodies and not express murine antibodies proteins, isimmunized to produce an antibody (e.g., as described inPCT/US2007/008231 and/or Lonberg et al., Nature 368 (1994): 856-859).Following immunization, antibody producing somatic cells (e.g., Blymphocytes) are fused with immortal cells, e.g., immortal myelomacells. Various methods for producing such fused cells (hybridomas) areknown in the art and described, for example, in Kohler and Milstein,Nature 256, 495-497, 1975. The hybridoma cells can then be culturedunder conditions sufficient for antibody production.

The present disclosure contemplates other methods for producingantibodies, e.g., ABL-MYC technology (as described, for example inLargaespada et al, Curr. Top. Microbiol. Immunol, 166, 91-96. 1990).

Library-Based Methods

The present disclosure also encompasses screening of libraries ofantibodies or proteins comprising antigen binding domains thereof (e.g.,comprising variable regions thereof) to identify a VEGF-B bindingantibody or protein comprising a variable region thereof.

Examples of libraries contemplated by this disclosure include naïvelibraries (from unchallenged subjects), immunized libraries (fromsubjects immunized with an antigen) or synthetic libraries. Nucleic acidencoding antibodies or regions thereof (e.g., variable regions) arecloned by conventional techniques (e.g., as disclosed in Sambrook andRussell, eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3,Cold Spring Harbor Laboratory Press, 2001) and used to encode anddisplay proteins using a method known in the art. Other techniques forproducing libraries of proteins are described in, for example in U.S.Pat. No. 6,300,064 (e.g., a HuCAL library of Morphosys AG); U.S. Pat.No. 5,885,793; U.S. Pat. No. 6,204,023; U.S. Pat. No. 6,291,158; or U.S.Pat. No. 6,248,516.

The proteins according to the disclosure may be soluble secretedproteins or may be presented as a fusion protein on the surface of acell, or particle (e.g., a phage or other virus, a ribosome or a spore).Various display library formats are known in the art. For example, thelibrary is an in vitro display library (e.g., a ribosome displaylibrary, a covalent display library or a mRNA display library, e.g., asdescribed in U.S. Pat. No. 7,270,969). In yet another example, thedisplay library is a phage display library wherein proteins comprisingantigen binding domains of antibodies are expressed on phage, e.g., asdescribed in U.S. Pat. No. 6,300,064; U.S. Pat. No. 5,885,793; U.S. Pat.No. 6,204,023; U.S. Pat. No. 6,291,158; or U.S. Pat. No. 6,248,516.Other phage display methods are known in the art and are contemplated bythe present disclosure. Similarly, methods of cell display arecontemplated by the disclosure, e.g., bacterial display libraries, e.g.,as described in U.S. Pat. No. 5,516,637; yeast display libraries, e.g.,as described in U.S. Pat. No. 6,423,538 or a mammalian display library.

Methods for screening display libraries are known in the art. In oneexample, a display library of the present disclosure is screened usingaffinity purification, e.g., as described in Scopes (In: Proteinpurification: principles and practice, Third Edition, Springer Verlag,1994). Methods of affinity purification typically involve contactingproteins comprising antigen binding domains displayed by the librarywith a target antigen (e.g., VEGF-B) and, following washing, elutingthose domains that remain bound to the antigen.

Any variable regions or scFvs identified by screening are readilymodified into a complete antibody, if desired. Exemplary methods formodifying or reformatting variable regions or scFvs into a completeantibody are described, for example, in Jones et al., J Immunol Methods.354:85-90, 2010; or Jostock et al., J Immunol Methods, 289: 65-80, 2004.Alternatively, or additionally, standard cloning methods are used, e.g.,as described in Ausubel et al (In: Current Protocols in MolecularBiology. Wiley Interscience, ISBN 047 150338, 1987), and/or (Sambrook etal (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratories, New York, Third Edition 2001).

Deimmunized, Chimeric, Humanized, Synhumanized, Primatized and HumanProteins

The proteins of the present disclosure may be a humanized protein.

The term “humanized protein” shall be understood to refer to a proteincomprising a human-like variable region, which includes CDRs from anantibody from a non-human species (e.g., mouse or rat or non-humanprimate) grafted onto or inserted into FRs from a human antibody (thistype of antibody is also referred to a “CDR-grafted antibody”).Humanized proteins also include proteins in which one or more residuesof the human protein are modified by one or more amino acidsubstitutions and/or one or more FR residues of the human protein arereplaced by corresponding non-human residues. Humanized proteins mayalso comprise residues which are found in neither the human antibody orin the non-human antibody. Any additional regions of the protein (e.g.,Fc region) are generally human. Humanization can be performed using amethod known in the art, e.g., U.S. Pat. No. 5,225,539, U.S. Pat. No.6,054,297, U.S. Pat. No. 7,566,771 or U.S. Pat. No. 5,585,089. The term“humanized protein” also encompasses a super-humanized protein, e.g., asdescribed in U.S. Pat. No. 7,732,578.

The proteins of the present disclosure may be human proteins. The term“human protein” as used herein refers to proteins having variable and,optionally, constant antibody regions found in humans, e.g. in the humangermline or somatic cells or from libraries produced using such regions.The “human” antibodies can include amino acid residues not encoded byhuman sequences, e.g. mutations introduced by random or site directedmutations in vitro (in particular mutations which involve conservativesubstitutions or mutations in a small number of residues of the protein,e.g. in 1, 2, 3, 4 or 5 of the residues of the protein). These “humanantibodies” do not necessarily need to be generated as a result of animmune response of a human, rather, they can be generated usingrecombinant means (e.g., screening a phage display library) and/or by atransgenic animal (e.g., a mouse) comprising nucleic acid encoding humanantibody constant and/or variable regions and/or using guided selection(e.g., as described in or U.S. Pat. No. 5,565,332). This term alsoencompasses affinity matured forms of such antibodies. For the purposesof the present disclosure, a human protein will also be considered toinclude a protein comprising FRs from a human antibody or FRs comprisingsequences from a consensus sequence of human FRs and in which one ormore of the CDRs are random or semi-random, e.g., as described in U.S.Pat. No. 6,300,064 and/or U.S. Pat. No. 6,248,516.

The proteins of the present disclosure may be synhumanized proteins. Theterm “synhumanized protein” refers to a protein prepared by a methoddescribed in WO2007/019620. A synhumanized protein includes a variableregion of an antibody, wherein the variable region comprises FRs from aNew World primate antibody variable region and CDRs from a non-New Worldprimate antibody variable region. For example, a synhumanized proteinincludes a variable region of an antibody, wherein the variable regioncomprises FRs from a New World primate antibody variable region and CDRsfrom a mouse or rat antibody.

The proteins of the present disclosure may be primatized proteins. A“primatized protein” comprises variable region(s) from an antibodygenerated following immunization of a non-human primate (e.g., acynomolgus macaque). Optionally, the variable regions of the non-humanprimate antibody are linked to human constant regions to produce aprimatized antibody. Exemplary methods for producing primatizedantibodies are described in U.S. Pat. No. 6,113,898.

In one example a protein of the disclosure is a chimeric protein. Theterm “chimeric proteins” refers to proteins in which an antigen bindingdomain is from a particular species (e.g., murine, such as mouse or rat)or belonging to a particular antibody class or subclass, while theremainder of the protein is from a protein derived from another species(such as, for example, human or non-human primate) or belonging toanother antibody class or subclass. In one example, a chimeric proteinis a chimeric antibody comprising a V_(H) and/or a V_(L) from anon-human antibody (e.g., a murine antibody) and the remaining regionsof the antibody are from a human antibody. The production of suchchimeric proteins is known in the art, and may be achieved by standardmeans (as described, e.g., in U.S. Pat. No. 6,331,415; U.S. Pat. No.5,807,715; U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397).

The present disclosure also contemplates a deimmunized protein, e.g., asdescribed in WO2000/34317 and WO2004/108158. De-immunized antibodies andproteins have one or more epitopes, e.g., B cell epitopes or T cellepitopes removed (i.e., mutated) to thereby reduce the likelihood that asubject will raise an immune response against the antibody or protein.

Other Proteins Comprising Antibody Variable Regions

The present disclosure also contemplates other proteins comprising avariable region or antigen binding domain of an antibody, such as:

-   (i) a single-domain antibody, which is a single polypeptide chain    comprising all or a portion of the V_(H) or a V_(L) of an antibody    (see, e.g., U.S. Pat. No. 6,248,516);-   (ii) diabodies, triabodies and tetrabodies, e.g., as described in    U.S. Pat. No. 5,844,094 and/or US2008152586;-   (iii) scFvs, e.g., as described in U.S. Pat. No. 5,260,203;-   (iv) minibodies, e.g., as described in U.S. Pat. No. 5,837,821;-   (v) “key and hole” bispecific proteins as described in U.S. Pat. No.    5,731,168;-   (vi) heteroconjugate proteins, e.g., as described in U.S. Pat. No.    4,676,980;-   (vii) heteroconjugate proteins produced using a chemical    cross-linker, e.g., as described in U.S. Pat. No. 4,676,980;-   (viii) Fab′-SH fragments, e.g., as described in Shalaby et al, J.    Exp. Med., 175: 217-225, 1992; or-   (ix) Fab₃ (e.g., as described in EP19930302894).

Constant Domain Fusions

The present disclosure encompasses a protein comprising a variableregion of an antibody and a constant region or Fc or a domain thereof,e.g., C_(H)2 and/or C_(H)3 domain. Suitable constant regions and/ordomains will be apparent to the skilled artisan and/or the sequences ofsuch polypeptides are readily available from publicly availabledatabases. Kabat et al also provide description of some suitableconstant regions/domains.

Constant regions and/or domains thereof are useful for providingbiological activities such as, dimerization, extended serum half-lifee.g., by binding to FcRn (neonatal Fc Receptor), antigen dependent cellcytotoxicity (ADCC), complement dependent cytotoxicity (CDC, antigendependent cell phagocytosis (ADCP).

The present disclosure also contemplates proteins comprising mutantconstant regions or domains, e.g., as described in U.S. Pat. No.7,217,797; U.S. Pat. No. 7,217,798; or US20090041770 (having increasedhalf-life) or US2005037000 (increased ADCC).

Stabilized Proteins

Neutralizing proteins of the present disclosure can comprise an IgG4constant region or a stabilized IgG4 constant region. The term“stabilized IgG4 constant region” will be understood to mean an IgG4constant region that has been modified to reduce Fab arm exchange or thepropensity to undergo Fab arm exchange or formation of a half-antibodyor a propensity to form a half antibody. “Fab arm exchange” refers to atype of protein modification for human IgG4, in which an IgG4 heavychain and attached light chain (half-molecule) is swapped for aheavy-light chain pair from another IgG4 molecule. Thus, IgG4 moleculesmay acquire two distinct Fab arms recognizing two distinct antigens(resulting in bispecific molecules). Fab arm exchange occurs naturallyin vivo and can be induced in vitro by purified blood cells or reducingagents such as reduced glutathione. A “half antibody” forms when an IgG4antibody dissociates to form two molecules each containing a singleheavy chain and a single light chain.

In one example, a stabilized IgG4 constant region comprises a proline atposition 241 of the hinge region according to the system of Kabat (Kabatet al., Sequences of Proteins of Immunological Interest Washington DCUnited States Department of Health and Human Services, 1987 and/or1991). This position corresponds to position 228 of the hinge regionaccording to the EU numbering system (Kabat et al., Sequences ofProteins of Immunological Interest Washington DC United StatesDepartment of Health and Human Services, 2001 and Edelman et al., Proc.Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue isgenerally a serine. Following substitution of the serine for proline,the IgG4 hinge region comprises a sequence CPPC. In this regard, theskilled person will be aware that the “hinge region” is a proline-richportion of an antibody heavy chain constant region that links the Fc andFab regions that confers mobility on the two Fab arms of an antibody.The hinge region includes cysteine residues which are involved ininter-heavy chain disulfide bonds. It is generally defined as stretchingfrom Glu226 to Pro243 of human IgG1 according to the numbering system ofKabat. Hinge regions of other IgG isotypes may be aligned with the IgG1sequence by placing the first and last cysteine residues forminginter-heavy chain disulfide (S-S) bonds in the same positions (see forexample WO2010/080538).

Additional Protein-Based VEGF-B Signaling Inhibitors

Other proteins that may interfere with the productive interaction ofVEGF-B with its receptor include mutant VEGF-B proteins.

In one example, the inhibitor is a soluble protein comprising one ormore domains of a VEGF-R1 that bind to VEGF-B (and, e.g., do notsubstantially bind to VEGF-A). In one example, the soluble proteinadditionally comprises a constant region of an antibody, such as an IgG1antibody. For example, the soluble protein additionally comprises a Fcregion and, optionally a hinge region of an antibody, e.g., an IgG1antibody.

In one example, the protein inhibitor is an antibody mimetic, e.g., aprotein scaffold comprising variable regions that bind to a targetprotein in a manner analogous to an antibody. A description of exemplaryantibody mimetics follows.

Immuno globulins and Immuno Globulin Fragments

An example of a compound of the present disclosure is a proteincomprising a variable region of an immunoglobulin, such as a T cellreceptor or a heavy chain immunoglobulin (e.g., an IgNAR, a camelidantibody).

Heavy Chain Immuno globulins

Heavy chain immunoglobulins differ structurally from many other forms ofimmunoglobulin (e.g., antibodies) in so far as they comprise a heavychain, but do not comprise a light chain. Accordingly, theseimmunoglobulins are also referred to as “heavy chain only antibodies”.Heavy chain immunoglobulins are found in, for example, camelids andcartilaginous fish (also called IgNAR).

The variable regions present in naturally occurring heavy chainimmunoglobulins are generally referred to as “V_(HH) domains” in camelidIg and V-NAR in IgNAR, in order to distinguish them from the heavy chainvariable regions that are present in conventional 4-chain antibodies(which are referred to as “V_(H) domains”) and from the light chainvariable regions that are present in conventional 4-chain antibodies(which are referred to as “V_(L) domains”).

Heavy chain immunoglobulins do not require the presence of light chainsto bind with high affinity and with high specificity to a relevantantigen. This means that single domain binding fragments can be derivedfrom heavy chain immunoglobulins, which are easy to express and aregenerally stable and soluble.

A general description of heavy chain immunoglobulins from camelids andthe variable regions thereof and methods for their production and/orisolation and/or use is found inter alia in the following referencesWO94/04678, WO97/49805 and WO 97/49805.

A general description of heavy chain immunoglobulins from cartilaginousfish and the variable regions thereof and methods for their productionand/or isolation and/or use is found inter alia in WO2005/118629.

V-Like Proteins

An example of a compound of the disclosure is a T-cell receptor. T cellreceptors have two V-domains that combine into a structure similar tothe Fv module of an antibody. Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991 describes how the two V-domains of the T-cell receptor(termed alpha and beta) can be fused and expressed as a single chainpolypeptide and, further, how to alter surface residues to reduce thehydrophobicity directly analogous to an antibody scFv. Otherpublications describing production of single-chain T-cell receptors ormultimeric T cell receptors comprising two V-alpha and V-beta domainsinclude WO1999/045110 or WO2011/107595.

Other non-antibody proteins comprising antigen binding domains includeproteins with V-like domains, which are generally monomeric. Examples ofproteins comprising such V-like domains include CTLA-4, CD28 and ICOS.Further disclosure of proteins comprising such V-like domains isincluded in WO1999/045110.

Adnectins

In one example, a compound of the disclosure is an adnectin. Adnectinsare based on the tenth fibronectin type III (¹⁰Fn3) domain of humanfibronectin in which the loop regions are altered to confer antigenbinding. For example, three loops at one end of the β-sandwich of the¹⁰Fn3 domain can be engineered to enable an Adnectin to specificallyrecognize an antigen. For further details see US20080139791 orWO2005/056764.

Anticalins

In a further example, a compound of the disclosure is an anticalin.Anticalins are derived from lipocalins, which are a family ofextracellular proteins which transport small hydrophobic molecules suchas steroids, bilins, retinoids and lipids. Lipocalins have a rigidβ-sheet secondary structure with a plurality of loops at the open end ofthe conical structure which can be engineered to bind to an antigen.Such engineered lipocalins are known as anticalins. For furtherdescription of anticalins see U.S. Pat. No. 7,250,297B1 orUS20070224633.

Affibodies

In a further example, a compound of the disclosure is an affibody. Anaffibody is a scaffold derived from the Z domain (antigen bindingdomain) of Protein A of Staphylococcus aureus which can be engineered tobind to antigen. The Z domain consists of a three-helical bundle ofapproximately 58 amino acids. Libraries have been generated byrandomization of surface residues. For further details see EP1641818.

Avimers

In a further example, a compound of the disclosure is an Avimer. Avimersare multidomain proteins derived from the A-domain scaffold family. Thenative domains of approximately 35 amino acids adopt a defined disulfidebonded structure. Diversity is generated by shuffling of the naturalvariation exhibited by the family of A-domains. For further details seeWO2002088171.

DARPins

In a further example, a compound of the disclosure is a Designed AnkyrinRepeat Protein (DARPin). DARPins are derived from Ankyrin which is afamily of proteins that mediate attachment of integral membrane proteinsto the cytoskeleton. A single ankyrin repeat is a 33 residue motifconsisting of two α-helices and a β-turn. They can be engineered to binddifferent target antigens by randomizing residues in the first α-helixand a β-turn of each repeat. Their binding interface can be increased byincreasing the number of modules (a method of affinity maturation). Forfurther details see US20040132028.

Methods for Producing Proteins Recombinant Expression

In the case of a recombinant protein, nucleic acid encoding same can becloned into expression vectors, which are then transfected into hostcells, such as E. coli cells, yeast cells, insect cells, or mammaliancells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells,human embryonic kidney (HEK) cells, or myeloma cells that do nototherwise produce an antibody. Exemplary cells used for expressing aprotein of the disclosure are CHO cells, myeloma cells or HEK cells.Molecular cloning techniques to achieve these ends are known in the artand described, for example in Ausubel et al., (editors), CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present) orSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (1989). A wide variety of cloning and in vitroamplification methods are suitable for the construction of recombinantnucleic acids. Methods of producing recombinant antibodies are alsoknown in the art. See U.S. Pat. No. 4,816,567 or U.S. Pat. No.5,530,101.

Following isolation, the nucleic acid is inserted operably linked to apromoter in an expression construct or expression vector for furthercloning (amplification of the DNA) or for expression in a cell-freesystem or in cells.

As used herein, the term “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences of agenomic gene, including the TATA box or initiator element, which isrequired for accurate transcription initiation, with or withoutadditional regulatory elements (e.g., upstream activating sequences,transcription factor binding sites, enhancers and silencers) that alterexpression of a nucleic acid, e.g., in response to a developmentaland/or external stimulus, or in a tissue specific manner. In the presentcontext, the term “promoter” is also used to describe a recombinant,synthetic or fusion nucleic acid, or derivative which confers, activatesor enhances the expression of a nucleic acid to which it is operablylinked. Exemplary promoters can contain additional copies of one or morespecific regulatory elements to further enhance expression and/or alterthe spatial expression and/or temporal expression of said nucleic acid.

As used herein, the term “operably linked to” means positioning apromoter relative to a nucleic acid such that expression of the nucleicacid is controlled by the promoter.

Many vectors for expression in cells are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, a sequence encoding an antibody (e.g.,derived from the information provided herein), an enhancer element, apromoter, and a transcription termination sequence. The skilled artisanwill be aware of suitable sequences for expression of an antibody.Exemplary signal sequences include prokaryotic secretion signals (e.g.,pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stableenterotoxin II), yeast secretion signals (e.g., invertase leader, afactor leader, or acid phosphatase leader) or mammalian secretionsignals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirusimmediate early promoter (CMV-IE), human elongation factor 1-α promoter(EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chainpromoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter(RSV), Adenovirus major late promoter, β-actin promoter; hybridregulatory element comprising a CMV enhancer/β-actin promoter or animmunoglobulin promoter or active fragment thereof. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture; baby hamster kidney cells(BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as forexample a yeast cell selected from the group comprising Pichia pastoris,Saccharomyces cerevisiae and S. pombe, include, but are not limited to,the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or theTEF1 promoter.

Means for introducing the isolated nucleic acid or expression constructcomprising same into a cell for expression are known to those skilled inthe art. The technique used for a given cell depends on the knownsuccessful techniques. Means for introducing recombinant DNA into cellsinclude microinjection, transfection mediated by DEAE-dextran,transfection mediated by liposomes such as by using lipofectamine(Gibco, Md., USA) and/or cellfectin (Gibco, Md., USA), PEG-mediated DNAuptake, electroporation and microparticle bombardment such as by usingDNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongstothers.

The host cells used to produce the antibody may be cultured in a varietyof media, depending on the cell type used. Commercially available mediasuch as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing mammalian cells. Media for culturingother cell types discussed herein are known in the art.

Protein Purification

Following production/expression, a protein of the disclosure is purifiedusing a method known in the art. Such purification provides the proteinof the disclosure substantially free of nonspecific protein, acids,lipids, carbohydrates, and the like. In one example, the protein will bein a preparation wherein more than about 90% (e.g. 95%, 98% or 99%) ofthe protein in the preparation is a protein of the disclosure.

Standard methods of peptide purification are employed to obtain anisolated protein of the disclosure, including but not limited to varioushigh-pressure (or performance) liquid chromatography (HPLC) and non-HPLCpolypeptide isolation protocols, such as size exclusion chromatography,ion exchange chromatography, hydrophobic interaction chromatography,mixed mode chromatography, phase separation methods, electrophoreticseparations, precipitation methods, salting in/out methods,immunochromatography, and/or other methods.

In one example, affinity purification is useful for isolating a fusionprotein comprising a label. Methods for isolating a protein usingaffinity chromatography are known in the art and described, for example,in Scopes (In: Protein purification: principles and practice, ThirdEdition, Springer Verlag, 1994). For example, an antibody or compoundthat binds to the label (in the case of a polyhistidine tag this may be,for example, nickel-NTA) is immobilized on a solid support. A samplecomprising a protein is then contacted to the immobilized antibody orcompound for a time and under conditions sufficient for binding tooccur. Following washing to remove any unbound or non-specifically boundprotein, the protein is eluted.

In the case of a protein comprising a Fc region of an antibody, proteinA or protein G or modified forms thereof can be used for affinitypurification. Protein A is useful for isolating purified proteinscomprising a human γ1, γ2, or γ4 heavy chain Fc region. Protein G isrecommended for all mouse Fc isotypes and for human γ3.

Nucleic Acid-Based VEGF-B Signaling Inhibitors

In one example of the disclosure, therapeutic and/or prophylacticmethods as described herein according to any example of the disclosureinvolve reducing expression of VEGF-B. For example, such a methodinvolves administering a compound that reduces transcription and/ortranslation of the nucleic acid. In one example, the compound is anucleic acid, e.g., an antisense polynucleotide, a ribozyme, a PNA, aninterfering RNA, a siRNA, a microRNA.

Antisense Nucleic Acids

The term “antisense nucleic acid” shall be taken to mean a DNA or RNA orderivative thereof (e.g., LNA or PNA), or combination thereof that iscomplementary to at least a portion of a specific mRNA molecule encodinga polypeptide as described herein in any example of the disclosure andcapable of interfering with a post-transcriptional event such as mRNAtranslation. The use of antisense methods is known in the art (see forexample, Hartmann and Endres (editors), Manual of Antisense Methodology,Kluwer (1999)).

An antisense nucleic acid of the disclosure will hybridize to a targetnucleic acid under physiological conditions. Antisense nucleic acidsinclude sequences that correspond to structural genes or coding regionsor to sequences that effect control over gene expression or splicing.For example, the antisense nucleic acid may correspond to the targetedcoding region of a nucleic acid encoding VEGF-B, or the 5′-untranslatedregion (UTR) or the 3′-UTR or combination of these. It may becomplementary in part to intron sequences, which may be spliced outduring or after transcription, for example only to exon sequences of thetarget gene. The length of the antisense sequence should be at least 19contiguous nucleotides, for example, at least 50 nucleotides, such as atleast 100, 200, 500 or 1000 nucleotides of a nucleic acid encodingVEGF-B. The full-length sequence complementary to the entire genetranscript may be used. The length can be 100-2000 nucleotides. Thedegree of identity of the antisense sequence to the targeted transcriptshould be at least 90%, for example, 95-100%.

Exemplary antisense nucleic acids against VEGF-B are described, forexample, in WO2003/105754.

Catalytic Nucleic Acid

The term “catalytic nucleic acid” refers to a DNA molecule orDNA-containing molecule (also known in the art as a “deoxyribozyme” or“DNAzyme”) or a RNA or RNA-containing molecule (also known as a“ribozyme” or “RNAzyme”) which specifically recognizes a distinctsubstrate and catalyzes the chemical modification of this substrate. Thenucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T(and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence forspecific recognition of a target nucleic acid, and a nucleic acidcleaving enzymatic activity (also referred to herein as the “catalyticdomain”). The types of ribozymes that are useful in this disclosure area hammerhead ribozyme and a hairpin ribozyme.

RNA Interference

RNA interference (RNAi) is useful for specifically inhibiting theproduction of a particular protein. Without being limited by theory,this technology relies on the presence of dsRNA molecules that contain asequence that is essentially identical to the mRNA of the gene ofinterest or part thereof, in this case an mRNA encoding a VEGF-B.Conveniently, the dsRNA can be produced from a single promoter in arecombinant vector host cell, where the sense and anti-sense sequencesare flanked by an unrelated sequence which enables the sense andanti-sense sequences to hybridize to form the dsRNA molecule with theunrelated sequence forming a loop structure. The design and productionof suitable dsRNA molecules for the present disclosure is well withinthe capacity of a person skilled in the art, particularly consideringWO99/32619, WO99/53050, WO99/49029, and WO01/34815.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides, such as at least 30 or 50nucleotides, for example at least 100, 200, 500 or 1000 nucleotides. Thefull-length sequence corresponding to the entire gene transcript may beused. The lengths can be 100-2000 nucleotides. The degree of identity ofthe sense and antisense sequences to the targeted transcript should beat least 85%, for example, at least 90% such as, 95-100%.

Exemplary small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. For example, the siRNA sequencecommences with the dinucleotide AA, comprises a GC-content of about30-70% (for example, 30-60%, such as 40-60% for example about 45%-55%),and does not have a high percentage identity to any nucleotide sequenceother than the target in the genome of the mammal in which it is to beintroduced, for example as determined by standard BLAST search.Exemplary siRNA that reduce expression of VEGF-B are commerciallyavailable from Santa Cruz Biotechnology or Novus Biologicals.

Short hairpin RNA (shRNA) that reduce expression of VEGF-B are alsoknown in the art and commercially available from Santa CruzBiotechnology.

Screening Assays

Compounds that inhibit VEGF-B signaling can be identified usingtechniques known in the art, e.g., as described below. Similarly,amounts of VEGF-B signaling inhibitors suitable for use in a methoddescribed herein can be determined or estimated using techniques knownin the art, e.g., as described below.

Neutralization Assays

For compounds that bind to VEGF-B and inhibit signaling, aneutralization assay can be used.

In one example, a neutralization assay involves contacting VEGF-B with acompound in the presence or absence of detectably labeled solubleVEGF-R1 or contacting detectably labeled VEGF-B with a compound in thepresence or absence of a cell expressing VEGF-R1 or a soluble VEGF-R1.The level of VEGF-B bound to the VEGF-R1 is then assessed. A reducedlevel of bound VEGF-B in the presence of the compound compared to in theabsence of the compound indicates the compound inhibits VEGF-B bindingto VEGF-R1 and, as a consequence VEGF-B signaling.

Another neutralization assay is described in WO2006/012688 and involvescontacting a fragment of VEGF-R1 comprising the second Ig-like domainimmobilized on a solid support with a subsaturating concentration ofrecombinant VEGF-B pre-incubated with a compound. Following washing toremove unbound protein, the immobilized protein is contacted withanti-VEGF-B antibody and the amount of bound antibody (indicative ofimmobilized VEGF-B) determined. A compound that reduces the level ofbound antibody compared to the level in the absence of the compound isconsidered an inhibitor of VEGF-B signaling.

In another example, a compound that inhibits VEGF-B signaling isidentified using a cell dependent on VEGF-B signaling for proliferation,e.g., a BaF3 cell modified as described in WO2006/012688 to express achimeric receptor incorporating the intracellular domain of the humanerythropoietin receptor and the extracellular domain of VEGF-R1. Cellsare cultured in the presence of VEGF-B and in the presence or absence ofa compound. Cell proliferation is then assessed using standard methods,e.g., colony formation assays, thymidine incorporation or uptake ofanother suitable marker of cell proliferation (e.g., a MTS dye reductionassay). A compound that reduces the level of proliferation in thepresence of VEGF-B is considered an inhibitor of VEGF-B signaling.

Compounds can also be assessed for their ability to bind to VEGF-B usingstandard methods. Methods for assessing binding to a protein are knownin the art, e.g., as described in Scopes (In: Protein purification:principles and practice, Third Edition, Springer Verlag, 1994). Such amethod generally involves labeling the compound and contacting it withimmobilized VEGF-B. Following washing to remove non-specific boundcompound, the amount of label and, as a consequence, bound compound isdetected. Of course, the compound can be immobilized and the VEGF-Blabeled. Panning-type assays can also be used. Alternatively, oradditionally, surface plasmon resonance assays can be used.

Expression Assays

A compound that reduces or prevents expression of VEGF-B is identifiedby contacting a cell with the compound and determining the level ofexpression of the VEGF-B. Suitable methods for determining geneexpression at the nucleic acid level are known in the art and include,for example, quantitative polymerase chain reaction (qPCR) or microarrayassays. Suitable methods for determining expression at the protein levelare also known in the art and include, for example, enzyme-linkedimmunosorbent assay (ELISA), fluorescence linked immunosorbent assay(FLISA), immunofluorescence or Western blotting.

In Vivo Assays

Compounds described herein can be tested for activity in animal models.In one example, the animal model is a model of metabolic dysfunction andhyperglycemia. For example, the diet-induced obese (DIO) mouse model.This is a well-established experimental paradigm and mice develop excessor ectopic lipid deposition in peripheral tissues which is associatedwith impaired insulin sensitivity and glucose uptake and the miceexhibit obesity, hyperinsulinemia, hyperglycemia, dyslipidemia andhypertension (Harberg, C. E., et al. Nature 490, 426-430; Collins, S.,et al. Pysiology & Behaviour 81, 243-248).

There are various known techniques for inducing an ischemic stroke in anon-human animal subject, such as, aorta/vena cava occlusion, externalneck torniquet or cuff, hemorrhage or hypotension, intracranialhypertension or common carotid artery occlusion, two-vessel occlusionand hypotension, four-vessel occlusion, unilateral common carotid arteryocclusion (in some species only), endothelin-1-induced constriction ofarteries and veins, middle cerebral artery occlusion (MCAO), spontaneousbrain infarction (in spontaneously hypertensive rats), macrosphereembolization, blood clot embolization or microsphere embolization.Hemorrhagic stroke can be modeled by infusion of collagenase into thebrain.

In one example, the model of stroke comprises middle cerebral arteryocclusion (MCAO) to produce an ischemic stroke as previously describedin Su et al Nature Medicine, 2008; 14: 731-737.

Pharmaceutical Compositions and Methods of Treatment

A compound that inhibits VEGF-B signaling (syn. active ingredient) isuseful for parenteral, topical, oral, or local administration, aerosoladministration, or transdermal administration, for prophylactic or fortherapeutic treatment. In one example, the compound is administeredparenterally, such as subcutaneously or intravenously.

Formulation of a compound to be administered will vary according to theroute of administration and formulation (e.g., solution, emulsion,capsule) selected. An appropriate pharmaceutical composition comprisingcompound to be administered can be prepared in a physiologicallyacceptable carrier. For solutions or emulsions, suitable carriersinclude, for example, aqueous or alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. A variety ofappropriate aqueous carriers are known to the skilled artisan, includingwater, buffered water, buffered saline, polyols (e.g., glycerol,propylene glycol, liquid polyethylene glycol), dextrose solution andglycine. Intravenous vehicles can include various additives,preservatives, or fluid, nutrient or electrolyte replenishers (See,generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed.1980). The compositions can optionally contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents and toxicityadjusting agents, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride and sodium lactate. The compoundcan be lyophilized for storage and reconstituted in a suitable carrierprior to use according to art-known lyophilization and reconstitutiontechniques.

The optimum concentration of the active ingredient(s) in the chosenmedium can be determined empirically, according to procedures known tothe skilled artisan, and will depend on the ultimate pharmaceuticalformulation desired.

The dosage ranges for the administration of the compound of thedisclosure are those large enough to produce the desired effect. Forexample, the composition comprises a therapeutically or prophylacticallyeffective amount of the compound.

As used herein, the term “effective amount” shall be taken to mean asufficient quantity of the compound to inhibit/reduce/prevent signalingof VEGF-B in a subject. The skilled artisan will be aware that such anamount will vary depending on, for example, the compound and/or theparticular subject and/or the type and/or the severity of stroke beingtreated. Accordingly, this term is not to be construed to limit thedisclosure to a specific quantity, e.g., weight or number of compounds.

As used herein, the term “therapeutically effective amount” shall betaken to mean a sufficient quantity of compound to reduce or inhibit oneor more symptoms of stroke.

As used herein, the term “prophylactically effective amount” shall betaken to mean a sufficient quantity of compound to prevent or inhibit ordelay the onset of one or more detectable symptoms of stroke.

In one example, the compound is administered in an amount effective tohave one or more of the following effects:

-   -   Reduce or prevent disruption of the blood brain barrier;    -   Reduce or prevent vascular permeability in the brain;    -   Reduce or prevent infarct size in the brain; and/or    -   Reduce or prevent intracranial hemorrhage.

The dosage should not be so large as to cause adverse side effects, suchas hyper viscosity syndromes, pulmonary edema, congestive heart failure,and the like. Generally, the dosage will vary with the age, condition,sex and extent of the disease in the patient and can be determined byone of skill in the art. The dosage can be adjusted by the individualphysician in the event of any complication.

Dosage can vary from about 0.1 mg/kg to about 300 mg/kg, e.g., fromabout 0.2 mg/kg to about 200 mg/kg, such as, from about 0.5 mg/kg toabout 20 mg/kg, in one or more dose administrations daily, for one orseveral days.

In some examples, the compound is administered at an initial (orloading) dose which is higher than subsequent (maintenance doses). Forexample, the compound is administered at an initial dose of betweenabout 1 mg/kg to about 30 mg/kg. The compound is then administered at amaintenance dose of between about 0.0001 mg/kg to about 1 mg/kg. Themaintenance doses may be administered every 7-35 days, such as, every 14or 21 or 28 days.

In some examples, a dose escalation regime is used, in which a compoundis initially administered at a lower dose than used in subsequent doses.This dosage regime is useful in the case of subject's initiallysuffering adverse events

In the case of a subject that is not adequately responding to treatment,multiple doses in a week may be administered. Alternatively, or inaddition, increasing doses may be administered.

In one example, the compound(s) of the disclosure is used in combinationwith at least one additional known compound which is currently beingused or is in development for preventing or treating stroke. Examples ofsuch known compounds include but are not limited to common thrombolyticagents such as tissue plasminogen activators (e.g. alteplase, reteplase,tenecteplase), anistreplase, streptokinase, urokinase, lanoteplase,desmoteplase and staphylokinase.

Additionally, the methods of the disclosure may also includeco-administration of at least one other therapeutic agent for thetreatment of another disease directly or indirectly related to stroke,including but not limited to: dyslipidemia, hypertension, obesity,neuropathy, and/or retinopathy, etc. Additional examples of agents thatcan be co-administered with the compound(s) according to the inventionare corticosteroids; immunosuppressive medications; antibiotics;antihypertensive and diuretic medications (such as ACE-inhibitors);lipid lowering agents such as bile sequestrant resins, cholestyramine,colestipol, nicotinic acid, and more particularly drugs and medicationsused to reduce cholesterol and triglycerides (e.g. fibrates (e.g.Gemfibrozil™) and HMG-CoA inhibitors such as Lovastatin™, Atorvastatin™,Fluvastatin™, Lescol™) Lipitor™, Mevacor™), Pravachol™, Pravastatin™,Simvastatin™, Zocor™, Cerivastatin™), etc); compounds that inhibitintestinal absorption of lipids (e.g. ezetiminde); nicotinic acid; andVitamin D.

As will be apparent from the foregoing, the present disclosure providesmethods of concomitant therapeutic treatment of a subject, comprisingadministering to a subject in need thereof an effective amount of afirst compound and a second compound, wherein said agent is a compoundof the disclosure (i.e., an inhibitor of VEGF-B signaling), and thesecond agent is for the prevention or treatment of stroke.

As used herein, the term “concomitant” as in the phrase “concomitanttreatment” includes administering a first agent in the presence of asecond agent. A concomitant therapeutic treatment method includesmethods in which the first, second, third or additional agents areco-administered. A concomitant therapeutic treatment method alsoincludes methods in which the first or additional agents areadministered in the presence of a second or additional agents, whereinthe second or additional agents, for example, may have been previouslyadministered. A concomitant therapeutic treatment method may be executedstep-wise by different actors. For example, one actor may administer toa subject a first agent and as a second actor may administer to thesubject a second agent and the administering steps may be executed atthe same time, or nearly the same time, or at distant times, so long asthe first agent (and/or additional agents) are after administration inthe presence of the second agent (and/or additional agents). The actorand the subject may be the same entity (e.g. a human).

The time of administration of an additional therapeutic agent can bemeasured from the time of administration of the compound of the presentinvention. The interval can be, for example, 5 minutes to 24 or 48hours. The interval may be for example, 15 min to 6 hours, 15 min to 4.5hours, 15 min to 3 hours, 15 min to 1 hour, 30 minutes to 6 hours, or 30min to 3 hours, or 30 min to 4.5 hours, or 1 to 3 hours, or 1 to 4.5hours, or 1 to 5 hours, or 1 to 6 hours or 1 to 7 hours, or 1 to 8hours, or 1 to 9 hours, or 1 to 10 hours.

In one example, the disclosure also provides a method for reducing aneffect of stroke or for treating stroke in a subject, the methodcomprising administering to the subject a thrombolytic agent, whereinthe subject is receiving or has previously received (e.g., since onsetof a symptom of stroke) an inhibitor of VEGF-B signaling to a subjectsuffering from stroke and receiving another treatment.

Kits

Another example of the disclosure provides kits containing compoundsuseful for the treatment of stroke as described above.

In one example, the kit comprises (a) a container comprising a compoundthat inhibits VEGF-B signaling as described herein and/or a thrombolyticcompound as described herein, optionally in a pharmaceuticallyacceptable carrier or diluent; and (b) a package insert withinstructions for reducing an effect of stroke in a subject.

In accordance with this example of the disclosure, the package insert ison or associated with the container. Suitable containers include, forexample, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds or contains a composition that is effective for treating thestroke and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is the compound that inhibits VEGF-B signaling. The label orpackage insert indicates that the composition is used for treating asubject eligible for treatment, e.g., one having or predisposed tostroke, with specific guidance regarding dosing amounts and intervals ofcompound and any other medicament being provided. The kit may furthercomprise an additional container comprising a pharmaceuticallyacceptable diluent buffer, such as bacteriostatic water for injection(BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrosesolution. The kit may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The present disclosure includes the following non-limiting Examples.

EXAMPLES Example 1 Increased Fatty Acid Uptake in Human Primary Brainderived Endothelial Cells In Vitro Reduced Glucose Uptake

Primary Endothelial Cells Stimulated with VEGF-B have Increased FattyAcid Uptake and Decreased Glucose Uptake

Human primary brain micro-vascular endothelial cells, HBMECs, (passagenumber <5) were cultured in 24-well plates in endothelial basal mediumwith complement pack including 5% fetal bovine serum (FCS) in a cellculture incubator at 37° C., 5% CO₂. Six hours prior to stimulation,endothelial cells were starved by replacing FCS with fatty acid freebovine serum albumin (FAF-BSA). Cells were stimulated by addition ofvehicle (Control), 100 ng/ml of VEGF-B₁₆₇ (B167), 100 ng/ml VEGF-B₁₈₆(B186) or 2 μg/ml anti-VEGF-B antibody 2H10 for 2 hours.

After stimulation, uptake of BODIPY-labelled fatty acids (FA) or afluorescent glucose analogue (2-NBDG) was measured. 2-NBDG was used as amarker of glucose uptake. Cells were incubated with BODIPY-C12 or 2-NBDGtracers for 5 or 20 minutes, respectively. Before adding the 2-NBDG, thecells were washed for 10 minutes with Kreb's Ringer buffer to removeculture media derived glucose. After incubation with FA or glucosetracers, cells were washed and fixed before image acquisition andquantification.

Stimulation with 100 ng/ml of either VEGF-B isoform (VEGF-B₁₆₇ orVEGF-B₁₈₆) significantly increased fatty acid (FA) uptake andsignificantly reduced glucose uptake. Treatment with the blocking VEGF-Bantibody 2H10 alone did not significantly alter fatty acid or glucoseuptake, yet a trend of decreased FA uptake and increased glucose uptakewas observed indicative of endogenous production and secretion of VEGF-Bin cultured endothelial cells.

These data show that cerebrovascular endothelial cells are able torespond to VEGF-B with increased FA uptake and decreased glucose uptake,suggesting that the uptake of lipids and glucose may be linked in areciprocal manner in brain endothelium.

FIGS. 1A and 1B show increased FA uptake and decreased glucose uptake inprimary brain derived endothelial cells treated with VEGF-B proteins.

Exposure of Primary Endothelial Cells to Fatty Acids Decreases GlucoseUptake

Human primary endothelial cells were grown in the presence or absence ofa mixture of sodium palmitate and sodium oleate (50 μM) for 2 h orovernight and thereafter subjected to the 2-NBDG tracer and assayed forglucose uptake.

Endothelial cells exposed to a FA rich environment and lipid loadingexhibit significantly decreased ability to take up glucose in vitro.This suggest that endothelial cells in a high lipid environment in vivo,such as in the diet-induced obesity (DIO) model, may respond similarlyand that this would translate into decreased tissue glucose uptake insettings of DIO in vivo.

FIG. 1C shows decreased glucose uptake in primary endothelial cellsexposed to fatty acids. Endothelial cells exposed to lipid loadingexhibit significantly decreased ability to take up glucose, suggestingthat exposure and uptake of FA in endothelial cells decreases glucoseuptake.

Example 2 Mice with Diet-Induced Obesity have Increased Blood GlucoseLevels and Increased Incidence of Ischemic Stroke

Mice with diet-induced obesity (DIO) have increased blood glucose levelsThree week old C57BL/6 mice were fed with high fat diet (60% caloriesfrom fat) or on a low fat control diet (normal chow, 10% calories fromfat) for 15 weeks. Blood glucose was measured at the same time of theday after withdrawal of the food for 2 h as a mean to stabilize theblood glucose levels. The tip of the tail was cut and a drop of bloodmeasured with a glucose meter.

DIO mice exhibited elevated blood glucose levels compared to age-matchedmice on normal chow, in accordance with previous studies (Hagberg et al.Nature 2012; Collins et al. Physiology & Behavior 2004).

FIG. 2A shows that mice with DIO have increased blood glucose levelscompared to age-matched mice on normal chow.

Mice with DIO have more Severe Strokes and Increased Incidence ofSpontaneous Hemorrhage and Larger Infarcts

Three week old male C57BL/6J mice were placed on a high fat diet (60%calories from fat) or on a low fat control diet (normal chow, 10%calories from fat), for a minimum of 12 weeks before inducing cerebralischemia (middle cerebral artery occlusion (MCAO) model).

To induce cerebral ischemia using the MCAO model, mice were anesthetizedwith chloral hydrate (450 mg/kg) and placed securely under a dissectingmicroscope. The left MCA was exposed by craniotomy, and a laser Dopplerflow probe placed on the surface of the cerebral cortex 1.5 mm dorsalmedian from the bifurcation of the MCA. The probe was connected to aflow meter and relative cerebral blood flow (CBF) determined from thetissue perfusion units (TPU) recorded with a continuous data acquisitionprogram. A 3.5-mW 540 nm laser was directed at the MCA from a distanceof 6 cm, and Rose Bengal dye (RB) (50 mg/kg), injected via the tailvein. Stable occlusion was achieved when the TPU drops to less than 20%of pre-occlusion levels and does not rebound within 10 min of laserwithdrawal.

To determine infarct volumes, brains were removed, cut into 2-mm thickcoronal sections and stained with 4% 2,3,5-triphenyltetrazolium-chloride(TTC). The areas of infarction at five coronal levels throughout thebrain were identified and infarct and hemispheric volumes measured.Infarct volumes were calculated by measuring and subtracting the volumeof the non-infarcted ipsilateral hemisphere from the volume of thecontralateral hemisphere.

Mice with DIO had more severe strokes than lean mice, with asignificantly increased incidence of spontaneous hemorrhage and largerinfarcts. Stroke outcomes were significantly worse in the DIO model thanin normal diet control mice, both in terms of infarct size, and anincreased incidence of spontaneous intracerebral hemorrhages (ICH).

FIGS. 2B and 2C shows that mice with DIO have larger stroke volume andincreased hemorrhage after ischemic stroke.

Mice with DIO have Significantly Increased Expression of the VEGF-BReceptor Nrp1 following Cerebral Ischemia

DIO mice were subjected to MCAO, as described above. At 3 hpost-ischemia, mice were perfused and sacrificed, and ipsi- andcontralateral hemispheres homogenized and total RNA prepared. Transcriptlevels of Vegfb and its receptors Vegfr1 (Flt1) and Neuropilin-1 (Nrp1)were quantified by qPCR.

Neither Vegfb nor Vegfr1 expression was significantly increased by 3 hof ischemia, relative to the non-ischemic contralateral hemisphere,however expression of Nrp1 was increased nearly 3-fold in the ischemichemisphere 3 h after MCAO, suggesting that VEGF-B signaling may increaselocally during cerebral ischemia.

FIG. 2D shows that Nrp1 expression, but not Vegf-b or Flt1 expression issignificantly increased in mice with DIO following ischemic stroke.

Example 3 Prophylactic Treatment with a Neutralizing Anti-VEGF-BAntibody (2H10) Prevents Progression of Ischemic Stroke in Mice with DIO

VEGF-B Antagonism Improves Glucose Uptake in Brains of Mice with DIO

C57BL/6J mice were fed normal chow (lean) or a high fat chow (DIO) andDIO mice were treated with either isotype control or anti-VEGF-B 2H10antibodies (16 mg/kg) twice a week for 30 weeks followed by [¹⁸F]-DG PETimaging.

DIO reduced glucose uptake (as determined using the labelled glucoseanalogue) in the brain compared to lean control mice, and long-terminhibition of VEGF-B in mice with DIO significantly improved glucoseuptake in the brain. These data suggest that similar to peripheraltissues, the systemic metabolic disturbances associated with DIO mayreduce the ability of the brain to take up glucose from the blood, andthat VEGF-B signaling may play a role in regulating CNS glucosemetabolism in the DIO model.

FIG. 3A shows VEGF-B antagonism with 2H10 improves glucose uptake in thebrains of mice with DIO.

Prophylactic Anti-VEGF-B Treatment Significantly Reduces the Severity ofIschemic Stroke in Mice with DIO

To directly test whether VEGF-B promotes neurovascular dysfunction andincreases the severity of ischemic stroke in a background of metabolicdisturbance, a thrombotic stroke was induced in mice with DIO, with andwithout treatment of the monoclonal antibody to VEGF-B, 2H10, or anisotype control.

Mice were treated for 1 week with 3 injections (I.P.) on days −7, −3,and 1 hour prior to MCAO with 16 mg/kg of either antibody. Mice werethen subjected to photothrombotic MCAO and analyzed as described above.Treatment with a VEGF-B blocking antibody, 2H10, in a DIO backgroundsignificantly reduces both infarct size and ICH.

FIGS. 3B and 3C shows prophylactic treatment with a VEGF-B blockingantibody (2H10) improves outcome after ischemic stroke in mice with DIO.

Mice with DIO Prophylactically Treated with Anti-VEGF-B RetainExpression of the Glucose Transporter Glut-1 following Cerebral Ischemia

Three week old C57BL/6J mice were fed high fat chow for 17 weeks, afterwhich they were pre-treated for 1 week, via IP injection on days −7, −3,and 1 hour prior to MCAO with 16 mg/kg of either isotype control oranti-VEGF-B 2H10 antibodies

DIO mice pre-treated with control or 2H10 antibodies were perfused andfixed in PFA at 1 h post-MCAO and brains subjected to vibratomesectioning. Sections were stained with antibodies directed againstGlut-1 and detected with immunofluorescence.

Staining for the glucose transporter Glut-1 in cerebral vessels of micewith DIO demonstrated that in the border of the ischemic zone, as earlyas 1 hour after MCAO, there was marked loss of Glut-1 staining comparedto similar vessels in the non-ischemic hemisphere (contra).

One week pre-treatment with the VEGF-B blocking antibody 2H10 prior toMCAO partially preserved Glut-1 antigen in vessels of the penumbra. Thissuggests that blocking VEGF-B signaling with 2H10 may promote betterglucose uptake by vessels in the ischemic penumbra by retaining Glut-1glucose transporter function and that this in turn enables vessels inthis region of diminished blood flow to better cope with the hypoxic andhypoglycemic environment of the penumbra.

FIG. 3D shows DIO mice pre-treated VEGF-B blocking antibody 2H10correlated with retained plasma membrane expression of Glut-1 in theischemic brain hemisphere compared with the non-ischemic contralateralhemisphere. Relative areas of immunofluorescence are plotted.

Prophylactic Anti-VEGF-B Treatment Reduces Blood-Brain-Barrier Leakagein Mice with DIO following Cerebral Ischemia

DIO mice were pre-treated with 2H10 or isotype control antibodies as inabove followed by MCAO. Immediately before induction of MCAO, mice wereinjected (IV) with a 70 kDa fluorescent dextran tracer to permitanalysis of the BBB permeability. At 1 hour after MCAO induction, themice were perfused and lightly post-fixated and brains analyzed for BBBpermeability.

As early as 1 hour after MCAO a dramatic loss of BBB integrity wasobserved in mice with DIO. This was significantly reduced by treatmentwith the VEGF-B blocking antibody 2H10. This suggests that blockingVEGF-B improves cerebrovascular function in mice with DIO during thefirst hour of ischemia.

FIG. 3E shows pre-treatment with the VEGF-B blocking antibody 2H10reduces blood-brain-barrier leakage 1 hour after ischemic stroke in micewith DIO.

Prophylactic Anti-VEGF-B Treatment Reduces Induction of OccludinPhosphorylation in Mice with DIO following Cerebral Ischemia

DIO mice were pre-treated for one week with 2H10 or isotype controlantibodies as above followed by MCAO. At 3 h post-ischemia, mice wereperfused and brains collected and immediately frozen. Cryo-sections wereobtained and subjected to immunofluorescent staining with aphosphoserine specific anti-Occludin antibody (serine residue 490) andfluorescence intensity quantified.

Occludin is a tight junction protein that is rapidly phosphorylatedafter MCAO and ischemia reperfusion injury (Muthusamy et al Journal ofcerebral blood flow and metabolism 2014). It has previously been shownthat phosphorylation of Occludin on Serine residue 490 is involved inBBB disruption and permeability (Murakami et al Diabetes 2012; Murakamiet al The Journal of Biological Chemistry 2009).

Isotype control (IgG) treated DIO mice showed increased Occludin serinephosphorylation (pS490) in the penumbra (ipsi), compared tocontralateral hemisphere (contra) 3 h after MCAO, indicating increasedendothelial tight junction disassembly and vascular permeability in theischemic hemisphere. Treatment with anti-VEGF-B antibody 2H10 diminishedinduction of Occludin phosphorylation in the ipsilateral hemispherepenumbra region supporting the notion that treatment with theanti-VEGF-B antibody 2H10 confers enhanced BBB integrity.

FIG. 3F shows anti-VEGF-B treatment diminishes Occludin phosphorylationfollowing ischemic stroke in mice with DIO.

Prophylactic Anti-VEGF-B Treatment Blocks Lipid Accumulation in CerebralBlood Vessels in Mice with DIO following Cerebral Ischemia

Since VEGF-B signaling down-regulates glucose uptake and increases lipiduptake (as shown above), cerebral vessels were examined for lipidaccumulation after MCAO in lean and obese mice, and in obese micetreated with the 2H10 antibody.

DIO mice were pre-treated with 2H10 or isotype control antibodies asdetailed above, followed by MCAO. DIO mice without pre-treatment andlean age-matched controls were also subjected to MCAO. At 3 hpost-ischemia, mice were perfused and brains collected and immediatelyfrozen. Cryo-sections were obtained, stained with an antibody to thelipid droplet coating protein, Adipophilin/Perilipin2 and quantified.

Within 3 h of MCAO significant uptake of lipids into cerebral vessels inthe ischemic hemisphere was observed. Relative to the non-ischemiccontralateral hemisphere, where very little adipophilin staining wasobserved, there was also significant uptake of lipids around vessel inthe ischemic hemisphere of lean mice. However, this was significantlyless than that seen in mice with DIO, and in these obese mice theincrease in perivascular lipid accumulation was largely blocked by 2H10treatment.

These data suggest that even in lean mice, MCAO rapidly induces lipiduptake, possibly to help meet metabolic demand in the ischemic tissue,and that DIO dramatically intensifies this response. Excessive andectopic lipid accumulation in the cerebrovascular bed might however beharmful, especially in the context of oxygen and glucose deprivationafter MCAO.

FIG. 3G shows that lipid accumulation in cerebral blood vessels afterischemic stroke is blocked by prophylactic treatment with theanti-VEGF-B antibody 2H10.

Example 4 Therapeutic Anti-VEGF-B Treatment in Combination with tPAThrombolysis Reduces Infarct Size, Hemorrhages and Increases Survival inMice with DIO

Mice with DIO were subjected to MCAO and treated 1 hr post stroke with2H10 or isotype control antibodies (16 mg/kg), as detailed above. Afteran additional 4 hrs, the mice received thrombolytic therapy byintravenous infusion of tPA (Alteplase, 10 mg/kg) to dissolve theinduced blood clots. Isolated brains of the surviving animals were thenanalyzed as described above.

Typically the efficacy of thrombolytic tPA treatment diminishes withtime, while the risk of hemorrhagic conversion rises (Ahmed et al LancetNeurology 2010). The present results show that 1 h delayed treatmentwith 2H10 after MCAO, followed by tPA thrombolysis 4 hrs later, reducedinfarct size, reduces hemorrhages, and prevents lethal hemorrhages asthe survival of the 2H10 treated animals was significantly highercompared to isotype treated controls that were subjected to late tPAthrombolysis.

The results suggest a therapeutic potential of VEGF-B antagonists instroke as a potential adjuvant therapy to tPA treatment that may improvethe safety of tPA thrombolysis by reducing hemorrhagic complicationsand/or may extend the treatment window for tPA. In a clinical setting,an extension of the therapeutic window is the most important steptowards treatment of more patients with thrombolytic therapy.

FIGS. 4A-C shows inhibition of VEGF-B with the VEGF-B blocking antibody2H10 improves outcomes after late thrombolysis.

1. A method of reducing an effect of stroke in a subject, the methodcomprising administering to the subject a compound that inhibits VEGF-Bsignaling.
 2. The method of claim 1, wherein the compound that inhibitsVEGF-B signaling is administered before or after the stroke, or isadministered before the stroke and is administered to a subject at riskof having a stroke.
 3. (canceled)
 4. The method of claim 1, wherein thesubject suffers from diabetes and/or obesity.
 5. The method of claim 4,wherein the diabetes is type 2 diabetes.
 6. The method of claim 1,wherein the subject has one or more of the following characteristics:has already suffered from a stroke and/or a transient ischemic attack;has a family history of stroke; suffers from heart disease; has highblood pressure; has high plasma low density lipoprotein levels; hasmetabolic syndrome has a cardiac abnormality; has undergone cardiacsurgery or hip replacement surgery.
 7. The method of claim 2, whereinthe compound is administered after the stroke and the method comprisesadministering a combination of the compound that inhibits VEGF-Bsignaling and a thrombolytic compound.
 8. The method of claim 7, whereinthe compound that inhibits VEGF-B signaling is administered before thethrombolytic compound.
 9. The method of claim 8, wherein administrationof the compound that inhibits VEGF-B signaling extends the time in whichthe thrombolytic compound can safely be administered to the subject. 10.The method of claim 7, wherein the thrombolytic compound is administeredmore than two hours after onset of symptoms of a stroke, or between twoand six hours after onset of symptoms of a stroke.
 11. (canceled) 12.The method of claim 7, wherein the subject has blood glucose levelsgreater than 400 mg/dL.
 13. The method of claim 7, wherein thethrombolytic compound is selected from the group consisting of a tissueplasminogen activator, lanetoplase, reteplase, staphylokinase,streptokinase, anistreplase, desmoteplase or an urokinase.
 14. Themethod of claim 1, wherein the compound(s) is(are) administered in anamount sufficient to have one or more of the following effects: reducinginfarct size in the subject; reducing hemorrhage score as assessed byintracerebral hemorrhage score in the subject; reducing incidence of ahemorrhage in a subject; reducing the likelihood of lethal hemorrhage ina subject; reducing brain edema in the subject following stroke; and/orreducing blood-brain-barrier breakdown or leakage in the subjectfollowing stroke.
 15. The method of claim 1, wherein the stroke is anischemic stroke.
 16. The method of claim 1, wherein the compound thatinhibits VEGF-B signaling binds to VEGF-B.
 17. The method of claim 16,wherein the compound is a protein comprising an antibody variable regionthat binds to or specifically binds to VEGF-B and neutralizes VEGF-Bsignaling.
 18. The method of claim 17, wherein the compound is a proteincomprising a Fv.
 19. The method of claim 18, wherein the protein isselected from the group consisting of: (i) a single chain Fv fragment(scFv); (ii) a dimeric scFv (di-scFv); or (iii) a diabody; (iv) atriabody; (v) a tetrabody; (vi) a Fab; (vii) a F(ab′)₂; (viii) a Fv;(ix) one of (i) to (ix) linked to a constant region of an antibody, Fcor a heavy chain constant domain (C_(H)) 2 and/or C_(H)3; or (x) anantibody.
 20. The method of claim 19, wherein the compound is: (i) aprotein comprising an antibody variable region that competitivelyinhibits the binding of antibody 2H10 (comprising a heavy chain variableregion (V_(H)) comprising a sequence set forth in SEQ ID NO: 2 and alight chain variable region (V_(L)) comprising a sequence set forth inSEQ ID NO: 3) to VEGF-B; or (ii) a protein comprising a humanized formof a variable region of antibody 2H10; or (iii) a humanized form ofantibody 2H10; or (iv) an antibody comprising a V_(H) comprising asequence set forth in SEQ ID NO: 5 and a V_(L) comprising a sequence setforth in SEQ ID NO:
 6. 21. (canceled)
 22. (canceled)
 23. The method ofclaim 1, wherein the compound that inhibits VEGF-B signaling inhibits orprevents expression of VEGF-B.
 24. The method of claim 23, wherein thecompound is selected from the group an antisense, a siRNA, a RNAi, aribozyme and a DNAzyme.